Reduction of lignin biosynthesis in transgenic plants

ABSTRACT

The present invention provides methods of selectively controlling lignin biosynthesis in plants such that lignification is reduced or enhanced, as desired. The invention provides, for example, a method of reducing lignification in a vascular plant by ectopically expressing a nucleic acid molecule encoding an AGL8-like gene product in the plant, whereby lignification is reduced due to ectopic expression of the nucleic acid molecule. An AGL8-like gene product useful in the invention can have, for example, substantially the amino acid sequence of an AGL8 ortholog such as  Arabidopsis  AGL8 (SEQ ID NO:2).

This application is a divisional application of U.S. application Ser.No. 09/339,998, filed Jun. 25, 1999, now U.S. Pat. No. 6,410,826, issuedJun. 25, 2002, which is based on, and claims the benefit of U.S.Provisional Application No. 60/090,649, filed Jun. 25, 1998, which isherein incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to agriculture and plant geneticengineering and more specifically to the production of geneticallymodified vascular plants in which the natural process of lignificationis reduced or enhanced.

BACKGROUND INFORMATION

Plant cell wall lignins (from the Latin lignum: wood) occur exclusivelyin higher plants and represent the second most abundant organic compoundon the earth's surface after cellulose, accounting for about 25% ofplant biomass. Cell wall lignification involves the deposition ofphenolic polymers (lignins) on the extracellular polysaccharide matrix.The polymers arise from the oxidative coupling of three cinnamylalcohols. The main function of lignins is to strengthen the plantvascular body, and the rigidity and structural support provided bylignification are thought to have had an important role in thesuccessful land colonization of plants. In addition, lignins providemechanical support for stems and leaf blades as well as resistance todiseases, insects, cold temperatures and other biotic and abioticstresses. Thus, lignification can be a beneficial process.

Although lignins are essential for competitive survival of vascularplants, their resistance to degradation has had a negative impact oncertain agricultural and industrial uses of plants. Animals lack theenzymes for degrading the polysaccharides in cell walls and depend onmicrobial fermentation to break down plant fibers. High ligninconcentration and methoxyl content reduce the digestibility of foragecrops, such as alfalfa, by cattle, with cattle able to digest only40-50% of legume fibers and 60-70% of grass fibers. Lignins are believedto limit forage digestibility by interfering with microbial degradationof fiber polysaccharides. However, small decreases in lignin content arepredicted to have a significant positive impact on forage digestibility.

High lignin content also is problematic in the wood products industries,which contribute about 4% of the US Gross National Product and are animportant component of the global economy. In wood-pulp and paperindustries, lignins are undesirable components that must be removed bycostly chemical pulping. Most of the lignin found in the space betweenthe fibers and in the secondary wall is removed during the pulping andbleaching process. The chemical treatments necessary to remove ligninsgenerate pollutants. Thus, both the digestibility of forage crops andthe pulping properties of trees are adversely effected by high lignincontent.

Genetic engineering has great promise for agriculture because it canaccelerate traditional breeding programs, cross reproductive barriersand introduce specific, desired traits. Genetic engineering can beparticularly advantageous to forestry because traditional methods arehampered by the long generation times of trees. Yet, previous attemptsto generate transgenic plants with altered lignin content have targetedbiosynthetic enzymes and resulted in undesirable pleiotropic effects.

Thus, there is a need for identifying genes that specifically regulatethe lignification process and for methods of genetically modifyingcultivated vascular plants to reduce their lignin content. Such methodswould allow the more efficient use of plant biomass in animal husbandrywhere lignin-containing grass and legume crops are used as forage and inthe pulp and paper industries. The present invention satisfies this needand provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a method of reducing lignification in avascular plant by ectopically expressing a nucleic acid moleculeencoding an AGL8-like gene product, whereby lignification is reduced dueto ectopic expression of the nucleic acid molecule. In a method of theinvention, the AGL8-like gene product can have substantially the aminoacid sequence of an AGL8 ortholog and can be, for example, ArabidopsisAGL8 (SEQ ID NO:2). The methods of the invention can be particularlyuseful for reducing lignification in woody plants such as Eucalyptus,leguminous forage crops such as alfalfa, and in forage grasses.

In one embodiment, the invention provides a method of reducinglignification by introducing into a vascular plant an exogenous nucleicacid molecule encoding an AGL8-like gene product to produce a transgenicvascular plant characterized by reduced lignification. In such a method,the exogenous nucleic acid molecule encoding an AGL8-like gene productcan be operatively linked to an exogenous regulatory element that is aconstitutive regulatory element or to a tissue-selective regulatoryelement, for example, an AGL1 regulatory element, an AGL5 regulatoryelement, or a lignified tissue-selective regulatory element such as afiber-selective regulatory element, xylem-selective regulatory elementor tracheid selective regulatory element.

The invention also provides a method of reducing lignification in avascular plant. The method includes the step of suppressing both AGL1and AGL5 expression in the vascular plant, whereby lignification isreduced.

Further provided by the invention is a transgenic vascular plantcharacterized by reduced lignification, which contains an ectopicallyexpressed nucleic acid molecule including a lignified tissue-selectiveregulatory element operatively linked to a nucleic acid moleculeencoding an AGL8-like gene product. The AGL8-like gene product can have,for example, substantially the amino acid sequence of an AGL8 orthologand can be, for example, Arabidopsis AGL8 (SEQ ID NO: 2). In atransgenic vascular plant of the invention characterized by reducedlignification, the lignified tissue-selective regulatory element can be,for example, a fiber-selective regulatory element, xylem-selectiveregulatory element or tracheid selective regulatory element. Tissuesderived from a transgenic vascular plant characterized by reducedlignification also are provided herein.

The invention also provides a method of enhancing lignification in avascular plant by ectopically expressing a nucleic acid moleculeencoding an AGL1/5-like gene product, whereby lignification is enhanceddue to ectopic expression of the nucleic acid molecule. In a method ofthe invention for enhancing lignification, the AGL1/5-like gene productcan have substantially the amino acid sequence of an AGL1 ortholog andcan be, for example, Arabdopsis AGL1 (SEQ ID NO:4). An AGL1/5-like geneproduct also can have, for example, substantially the amino acidsequence of an AGL5 ortholog and can be, for example, Arabdopsis AGL5(SEQ ID NO: 6). The methods of the invention can be particularlyvaluable for enhancing lignification in woody plants or trees that areproduced for direct utilization as fuel.

In one embodiment, the invention provides a method of enhancinglignification in a vascular plant by introducing an exogenous nucleicacid molecule encoding an AGL1/5-like gene product into the vascularplant to produce a transgenic vascular plant characterized by enhancedlignification. The exogenous nucleic acid molecule encoding anAGL1/5-like gene product can be operatively linked to an exogenousregulatory element, which can be a constitutive regulatory element ortissue-selective regulatory element. An AGL1/5-like gene product usefulin the invention can have substantially the amino acid sequence of anAGL1 ortholog such as Arabdopsis AGL1 (SEQ ID NO:4), or can havesubstantially the amino acid sequence of an AGL5 ortholog such asArabdopsis AGL5 (SEQ ID NO:6).

The invention additionally provides methods of enhancing lignificationin a vascular plant by suppressing AGL8-like gene product expression inthe vascular plant, whereby lignification is enhanced.

Further provided by the invention is a transgenic vascular plantcharacterized by enhanced lignification, comprising an ectopicallyexpressed nucleic acid molecule comprising a lignified tissue-selectiveregulatory element operatively linked to a nucleic acid moleculeencoding an AGL1/5-like gene product. In a transgenic vascular plant ofthe invention characterized by enhanced lignification, the AGL1/5-likegene product can have substantially the amino acid sequence of an AGL1ortholog such as Arabdopsis AGL1 (SEQ ID NO:4), or substantially theamino acid sequence of an AGL5 ortholog such as Arabdopsis AGL5 (SEQ IDNO:6), and the lignified tissue-selective regulatory element can be, forexample, a fiber-selective regulatory element, xylem-selectiveregulatory element or a tracheid selective regulatory element. Tissuesderived from a transgenic vascular plant of the invention characterizedby enhanced lignification also are provided.

The invention also provides kits for producing a transgenic vascularplant characterized by altered lignification. Such kits contain anucleic acid molecule including a lignified tissue-selective regulatoryelement and a nucleic acid molecule encoding an AGL8-like gene product,AGL1-like gene product or AGL5-like gene product. Lignifiedtissue-selective regulatory elements useful in a kit of the inventioninclude xylem-selective regulatory elements, tracheid-selectiveregulatory elements, and fiber-selective regulatory elements.

The invention also provides methods of enhancing lignification in avascular plant by ectopically expressing a nucleic acid moleculeencoding an R-like bHLH gene product in the vascular plant, wherelignification is enhanced due to ectopic expression of the nucleic acidmolecule. In a method of the invention, the R-like bHLH gene product canhave substantially the amino acid sequence of an R-like bHLH orthologsuch as SEQ ID NO:25. Such methods can be particularly useful forenhancing lignification in woody plants such as trees produced fordirect utilization as fuel.

In one embodiment, the invention provides a method of enhancinglignification by introducing an exogenous nucleic acid molecule encodinga R-like bHLH gene product into a vascular plant to produce a transgenicvascular plant characterized by enhanced lignification. The exogenousnucleic acid molecule encoding a R-like bHLH gene product can beoperatively linked to an exogenous regulatory element such as aconstitutive regulatory element or tissue-selective regulatory element.

The invention also provides a transgenic vascular plant characterized byenhanced lignification, which contains an ectopically expressed nucleicacid molecule including a heterologous regulatory element operativelylinked to a nucleic acid molecule encoding a R-like bHLH gene product.The encoded R-like bHLH gene product can have substantially the aminoacid sequence of a R-like bHLH ortholog such as the Arabdopsis orthologSEQ ID NO:25.

The invention further provides a method of reducing lignification in avascular plant by suppressing R-like bHLH expression in said vascularplant, whereby lignification is reduced. In addition, the inventionprovides a non-naturally occurring vascular plant characterized byreduced lignification, in which R-like bHLH expression in suppressed,whereby lignification is reduced. In one embodiment, the non-naturallyoccurring vascular plant does not have suppressed R-like bHLH expressiondue to ectopic expression of AGL8 or due to suppressed AGL1 and AGL5expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cell types of the Arabdopsis fruit at maturity.Indicated are the two lignified cell types: the lignified valve marginand the lignified fifth valve layer (“lignified layer”).

FIG. 2 shows characterization of the lignification pattern of wild-typeand agl8 (“ful”) fruits. Transverse sections of wild-type and agl8fruits (stage 17) were stained with toluidine blue and viewed withNomaraki optics. Bright field (photos on left) and dark fieldphotographs (photos on right) are shown of the same wild type (topphotos) and agl8 (bottom photos) sections. The staining andautofluorescence patterns of lignified cells differ between wild-type(top photos) and agl8 (bottom photos) sections. Whereas wild-typesections show patches of lignified cells adjacent to the valve margin,and lignification of the fifth valve cell layer, agl8 sections showlignification of additional valve cell layers corresponding to mesophylllayers. Lignification of the fifth valve cell layer and of the vascularbundles in the replum appears unaffected in agl8 fruits, although thevascular bundles appear more disorganized.

FIG. 3 shows histological and molecular characterization of wild-type(“wt”) and agl1 agl5 (“shp1 shp2”) fruits. (A-D) Transverse sections ofwild type and agl1 agl5 fruits (stage 17) were stained with toluidineblue and viewed with Nomarski optics. Dark field (A,B) and bright-fieldphotographs (C,D) are shown of the same wild type (A,C) and agl1 agl5(B,D) sections. The autofluorescence pattern of lignified cells differsbetween wild-type and agl1 agl5 sections at the valve margin. Whereasboth wild-type and agl1 agl5 sections exhibit lignification of thevascular bundles (vb) at either ends of the replum and of the fifthvalve cell layer (vl), in wild-type fruits small patches of valve margin(vm) cells immediately adjacent to the dehiscence zone are alsolignified and these lignified cells are absent in agl1 agl5 fruits.Positions of the lignified valve margin cells, valve layer and vascularbundles are also indicated in the wild-type bright-field photograph.(E-H) Expression of valve margin molecular markers in wild-type and agl1agl5 fruits (stage 17). Transverse sections of wild-type (E,G) and agl1agl5 (F,H) fruits containing either the GT140 (E,F) or YJ36 (G,H)molecular markers are shown. In wild-type fruits (E), expression of theGT140 marker occurs in stripes at the valve margin (vm) and thesestripes are absent in agl1 agl5 fruits (F). The YJ36 marker is expressedon the outer and inner surfaces of the valve margin in wild-type (G) butnot in agl1 agl5 (H) fruits. YJ36 expression is also found within theseptum (s) of both wild-type and agl1 agl5 fruits. All scale barsrepresent 100 um.

FIG. 4 shows characterization of 35S::AGL1 35S::AGL5 fruits. (A)Photograph of 35S::AGL1 35S::AGL5 fruit near maturity (stage 17). As inagl8 fruits, the valves (v) of 35S::AGL1 35S::AGL5 fruits usually tearopen due to seed crowding. (B) Scanning electron micrograph of the35S::AGL1 35S::AGL5 fruit (stage 17). Guard cells and associatedsubsidiary cells are not apparent in 35S::AGL1 35S::AGL5 valves. Insteadthe valve epidermis consists of a homogeneous population of long,slender cells. (C,D) Transverse sections of wild-type (C) and 35S::AGL135S::AGL5 fruits (D) show ectopic lignification of all valve mesophyllcells (me) rather than just the valve margin (vm) cells adjacent to thedehiscence zone as found in wild-type fruits (C). Lignification of thevascular bundles (vb) and of the fifth valve layer (vl) is not affectedin 35S::AGL1 35S::AGL5 fruits. Carpelloid sepals (cs) present in theouter whorl of 35S::AGL1 35S::AGL5 flowers (A,D) also show some ectopiclignification. All scale bars represent 100 μm.

FIG. 5 shows the three cinnamyl alcohols and their principal bondingpatterns in native lignins. A. Structure of three cinnamyl alcohols.Structure of (1) p-coumaryl alcohol; (2) coniferyl alcohol; and (3)sinapyl alcohol. B. Principal bonding patterns between phenolic units innative lignins. (a) guaiacylglycerol-β-aryl ether; (b) phenylcoumaran;(c) diarylpropane; (d) resinol; (e) biphenyl; and (f) diphenyl ether.The pino-, medio-, and syringa-resinol structures involve 2G, 1G/1S, and2S units. R is H or OCH₃.

FIG. 6 shows expression of the GT140 valve margin marker (R-like bHLH)in wild-type, agl1 agl5 (“shp1 shp2”), agl8 (“ful”), and agl1 agl5 agl8(“shp1 shp2 ful”) fruits. Transverse sections of wild-type, agl1 agl5,agl8, and agl1 agl5 agl8 fruits (stage 16/17) containing the GT140molecular marker are shown. In wild-type fruits (A), expression of theGT140 marker occurs in stripes at the valve margin, and these stripesare largely absent in agl1 agl5 fruits (B). Expression of the GT140marker expands throughout the valves of agl8 fruits (C) and agl1 agl5agl8 fruits (D), although expression of the marker appears qualitativelyweaker in fruits of the triple mutant.

FIG. 7 shows the nucleotide (SEQ ID NO:1) and amino acid (SEQ ID NO:2)sequence of Arabdopsis AGL8.

FIG. 8 shows the nucleotide sequence (SEQ ID NO:24) and amino acid (SEQID NO:25) of Arabdopsis R-like basic helix-loop-helix transcriptionfactor (R-like bHLH). The nucleotide sequence SEQ ID NO:24 includessufficient promoter sequence to give valve margin specific expression.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the surprising discovery that theAGL8, AGL1, AGL5 and the R-like basic helix-loop-helix transcriptionfactor (R-LIKE bHLH) regulate the process of lignification. As disclosedherein, an agl8 mutant Arabdopsis plant exhibits altered lignificationproperties, displaying enhanced lignification. Whereas wild type plantsshow patches of lignified cells adjacent to the valve margin, andlignification of the fifth valve cell layer, agl8 plants exhibitlignification of additional valve cell layers corresponding to internalmesophyll layers (see FIGS. 1 and 2). Furthermore, in a transgenicvascular plant constitutively expressing AGL8 under control of thecauliflower mosaic virus (35S CaMV) promoter, the number of lignifiedcells adjacent to the dehiscence zone was reduced. These resultsindicate that, in nature, the AGL8 transcription factor is a negativeregulator of lignification.

As further disclosed herein, the AGL1, AGL5 and R-Like bHLHtranscription factors can be positive regulators of lignification. Asshown in FIG. 3, patches of lignified cells adjacent to the dehiscencezones in wild type plants were absent in fruit from an agl1 agl5 doublemutant Arabdopsis plant although lignification of the fifth valve celllayer was not affected. As shown in FIG. 4, transgenic 35S::AGL1 and35S::AGL1 35S::AGL5 Arabdopsis lines were characterized by enhancedlignification, in particular ectopic lignification of the valvemesophyll layers with the most extensive lignification apparent in35S::AGL1 35S::AGL5 fruits.

As disclosed herein, the R-like bHLH gene product also can be a positiveregulator of lignification: R-like bHLH is ectopically expressed in allvalve cell layers of agl8 and 35S::AGL1 35S::AGL5 fruits, which arecharacterized by enhanced lignification. Furthermore, R-like bHLH waslargely absent from cells at the valve margin in agl1 agl5 fruitcharacterized by reduced lignification. Together, the results disclosedherein indicate that the natural balance of AGL8, AGL1, AGL5, and R-likebHLH, each of which are referred to herein as a “lignificationregulatory factor,” can be used to reduce or enhance lignification,thereby providing improved vascular plant varieties for human use.

Thus, the present invention provides a method of reducing lignificationin a vascular plant by ectopically expressing a nucleic acid moleculeencoding an AGL8-like gene product, whereby lignification is reduced dueto ectopic expression of the nucleic acid molecule. In a method of theinvention, the AGL8-like gene product can have substantially the aminoacid sequence of an AGL8 ortholog and can be, for example, ArabdopsisAGL8 (SEQ ID NO:2). The methods of the invention can be particularlyuseful for reducing lignification in a woody plant such as Eucalyptus,cottonwood, alder, Douglas fir, Hemlock, pine or spruce. The methods ofthe invention also are valuable for reducing lignification in aleguminous plant, for example, a leguminous forage crop such as alfalfa,clover, lucerne, birdsfoot trefoil, Stylosanthes, Lotononis bainessii orsainfoin. Similarly, the methods of the invention can be used to reducelignification in a forage grass such as bahiagrass, bermudagrass,dallisgrass, pangolagrass, big bluestem, indiangrass, switchgrass,smooth bromegrass, orchardgrass, timothy, Kentucky bluegrass or tallfescue.

In one embodiment, the invention provides a method of reducinglignification by introducing into a vascular plant an exogenous nucleicacid molecule encoding an AGL8-like gene product to produce a transgenicvascular plant characterized by reduced lignification. In such a method,the exogenous nucleic acid molecule encoding an AGL8-like gene productcan be operatively linked to an exogenous regulatory element that is aconstitutive regulatory element or to a tissue-selective regulatoryelement, for example, an AGL1 regulatory element, an AGL5 regulatoryelement, or a lignified tissue-selective regulatory element such as afiber-selective regulatory element, xylem-selective regulatory elementor tracheid selective regulatory element.

Lignins represent the second most abundant organic compound on theearth's surface after cellulose and account for about 25% of the plantbiomass. Lignins are typically associated with the development of thevascular system in plants. Lignins are present in a various cell types,with the greatest proportion of lignins deposited in cells walls oftracheids, vessel elements, xylem and phloem fibers, and sclereids, withthe nature of the lignins differing according to cell type.

Lignins are amorphous heteropolymers that are produced by the oxidativecoupling of the three cinnamyl alcohols, p-coumaryl, coniferyl, andsinapyl alcohol, producing, respectively, H (hydroxyphenyl), G(guaiacyl) and S (syringyl) units in the lignin polymer (see FIG. 5A).Lignins exhibit a high degree of structural variability, which isdependent upon the species of origin and the tissue and cell types. Thisheterogeneity is principally reflected in the relative proportion of thethree constituent monomers, the different types of interunit linkagesand the occurrence of nonconventional phenolic units within the polymer(Barceló, International Rev. Cytology 176:87-132 (1997), which isincorporated herein by reference). Distinctive variation in lignincontent is found between the gymnosperms and angiosperms. Ingymnosperms, lignins are typically composed of G units with a minorproportion of H units, while in angiosperms lignin is mainly composed ofG-S units.

The three cinnamyl alcohols are oxidatively coupled to form ahydrophobic network of phenylpropanoid units. Phenylpropane units areinterconnected in lignins by a series of ether and carbon-carbonlinkages, in various bonding patterns, leading to several mainsubstructures: guaiacylglycerol-β-aryl-ether, phenylcoumaran,diarylpropane, resinol, biphenyl, and diphenyl ether (see FIG. 5B).

The most frequent inter-unit bonds, β-O-4, are present inguaiacylglycerol-β aryl ether substructures and are the targets of mostlignin depolymerization processes. In contrast, other bonds, such as β-5(in phenylcoumaran), β-1 (in diarylpropane); β-β (in resinol), 5-5 (inbiphenyl), and, 5-O-4 (in diphenyl ether) interunit bonds, are veryresistant to degradation. In addition to these main inter-unit linkages,there are minor ones, such as the β-6 bonds of phenylisochromanstructures, or the noncyclic benzyl ether bonds, α-O-4.

Biosynthesis of lignins begins with enzymes in the phenylpropanoidpathway, phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroyxylase(C4H), p-coumarate-3-hydroxylase (C3H), O-methyltransferase (OMT),ferulate-5-hydroxylase (F5H), and hydroxylcinnamate CoA ligase (4CL).The end products of this pathway, the hydroxycinnamoyl CoAs, are theprecursors of lignins but also of other phenolic compounds thataccumulate in great amounts in plant tissues such as flavanoids andtannins.

The lignin-specific pathway involves two reductive steps that convertthe hydroxycinnamoyl-CoA esters into hydroxycinnamyl alcohols. These twoconsecutive steps are catalyzed by the enzymes cinnamoyl-CoA reductase(CCR) and cinnamyl alcohol dehydrogenase (CAD), which are consideredspecific to the lignification pathway. In particular, CCR catalyzes theconversion of hydroxycinnamoyl-CoA esters to their correspondingaldehydes as the first step in the lignin-specific pathway. CAD appearsas a polymorphic enzyme in angiosperms and apparently a single enzyme ingymnosperms (Lacombe et al., The Plant J. 11:429-441 (1997), which isincorporated by reference herein).

As used herein, the term “lignification” refers to the process givingrise to a polymer containing one or more of the H (hydroxyphenyl), G(guaiacyl) or S (syringyl) units. The H, G, and S units can be coupledby an ether, carbon-carbon, or other linkage; can be linear or branched;and can vary in the extent of their methylation. In addition, the term“lignification,” as used herein, refers to the production of relativelysmall lignins such as lignans and neolignans, which are products thatgenerally result from the oxidative coupling of two cinnamyl alcohols(or cinnamic acids) although other oligomeric forms can exist. Lignansare phenylpropanoid units interconnected via β-β carbon-carbon linkagesand, in this bonding pattern, differ from neolignans, which areinterconnected via linkages other than β-β linkages. Thus, the term“lignification” is used herein to refer to the formation of naturallyoccurring and non-naturally occurring lignins as well as lignans,neo-lignans and other lignin-like compounds.

The term “ectopically,” as used herein in reference to expression of anucleic acid molecule encoding a lignification regulatory factor such asan AGL8-like gene product, refers to an expression pattern that isdistinct from the expression pattern in a wild type vascular plant.Thus, one skilled in the art understands that ectopic expression of anucleic acid encoding, for example, an AGL8-like gene product can referto expression in a cell type other than a cell type in which the nucleicacid molecule normally is expressed, or at a time other than a time atwhich the nucleic acid molecule normally is expressed, or at a levelother than the level at which the nucleic acid molecule normally isexpressed. In wild type Arabidopsis, for example, AGL8 expression isnormally restricted during the later stages of floral development to thecarpel valves and is not seen in the replum, which is the small strip ofcells separating the carpel valves. However, under control of aconstitutive promoter such as the cauliflower mosaic virus 35S promoter,AGL8 is expressed in the replum and, additionally, is expressed athigher than normal levels in other tissues such as valve margin and,thus, is ectopically expressed.

The term “reduced,” as used herein in reference to lignification in anon-naturally occurring vascular plant of the invention, means asignificantly decreased extent of lignification in one or more tissuesas compared to the extent of lignification in a corresponding wild typevascular plant. Thus, the term “reduced” is used broadly to encompassboth lignification that is significantly diminished as compared to thelignification in a wild type vascular plant as well as the absence oflignification. The term “reduced” also encompasses lignification that issignificantly decreased in one or more tissues while wild type levels oflignification persist elsewhere in the vascular plant. One skilled inthe art understands that the term “reduced” refers to a steady statelevel of lignification and encompasses both decreased synthesis andincreased degradation of lignins.

It is recognized that there can be natural variation in the extent oflignification within a vascular plant species or variety. However,“reduced” lignification in a non-naturally occurring vascular plant ofthe invention readily can be identified by sampling a population of thenon-naturally occurring vascular plants and determining that the extentof lignification is significantly decreased, on average, as compared tothe normal distribution of lignification in a population of thecorresponding wild type plant species or variety. Thus, production ofnon-naturally occurring vascular plants of the invention provides ameans to skew the normal distribution of the extent of lignification.

As used herein, the term “non-naturally occurring,” when used inreference to a vascular plant, means a vascular plant that has beengenetically modified by man. A transgenic vascular plant of theinvention, for example, is a non-naturally occurring vascular plant thatcontains an exogenous nucleic acid molecule encoding a lignificationregulatory factor such as an AGL8-like gene product and, therefore, hasbeen genetically modified by man. In addition, a vascular that containsa mutation in, for example, an AGL8-like gene product regulatory elementor coding sequence as a result of calculated exposure to a mutagenicagent, such as a chemical mutagen, or an “insertional mutagen,” such asa transposon, also is considered a non-naturally occurring plant, sinceit has been genetically modified by man. In contrast, a vascular plantcontaining only spontaneous or naturally occurring mutations is not a“non-naturally occurring vascular plant” as defined herein and,therefore, is not encompassed within the invention. One skilled in theart understands that, while a non-naturally occurring vascular planttypically has a nucleotide sequence that is altered as compared to anaturally occurring vascular plant, a non-naturally occurring vascularplant also can be genetically modified by man without altering itsnucleotide sequence, for example, by modifying its methylation pattern.

The present invention relates to the use of nucleic acid moleculesencoding particular “AGAMOUS-LIKE” or “AGL” gene products. AGAMOUS (AG)is a floral organ identity gene, one of a related family oftranscription factors that, in various combinations, specify theidentity of the floral organs: the petals, sepals, stamens and carpels(Bowman et al., Devel. 112:1-20 (1991); Weigel and Meyerowitz, Cell78:203-209 (1994); Yanofsky, Annual Rev. Plant Physiol. Mol. Biol.46:167-188 (1995)). The AGAMOUS gene product is essential forspecification of carpel and stamen identity (Bowman et al., The PlantCell 1:37-52 (1989); Yanofsky et al., Nature 346:35-39 (1990)). Relatedgenes have recently been identified and denoted “AGAMOUS-LIKE” or “AGL”genes (Ma et al., Genes Devel. 5:484-495 (1991); Mandel and Yanofsky,The Plant Cell 7:1763-1771 (1995), which is incorporated herein byreference).

AGL8, like AGAMOUS and other AGL genes, is characterized, in part, inthat it is a plant MADS box gene. The plant MADS box genes generallyencode proteins of about 260 amino acids including a highly conservedMADS domain of about 56 amino acids (Riechmann and Meyerowitz, Biol.Chem. 378:1079-1101 (1997), which is incorporated herein by reference).The MADS domain, which was first identified in the Arabdopsis AGAMOUSand Antirrhimum majus DEFICIENS genes, is conserved among transcriptionfactors found in humans (serum response factor; SRF) and yeast (MCM1;Norman et al., Cell 55:989-1003 (1988); Passmore et al., J. Mol. Biol.204:593-606 (1988), and is the most highly conserved region of the MADSdomain proteins. The MADS domain is the major determinant of sequencespecific DNA-binding activity and can also perform dimerization andother accessory functions (Huang et al., The Plant Cell 8:81-94 (1996)).The MADS domain frequently resides at the N-terminus, although someproteins contain additional residues N-terminal to the MADS domain.

The “intervening domain” or “I-domain,” located immediately C-terminalto the MADS domain, is a weakly conserved domain having a variablelength of approximately 30 amino acids (Purugganan et al., Genetics140:345-356 (1995)). In some proteins, the I-domain plays a role in theformation of DNA-binding dimers. A third domain present in plant MADSdomain proteins is a moderately conserved 70 amino acid region denotedthe “keratin-like domain” or “K-domain.” Named for its similarity toregions of the keratin molecule, the structure of the K-domain appearscapable of forming amphipathic helices and may mediate protein-proteininteractions (Ma et al., Genes Devel. 5:484-495 (1991)). The mostvariable domain, both in sequence and in length, is the carboxy-terminalor “C-domain” of the MADS domain proteins. Dispensable for DNA bindingand protein dimerization in some MADS domain proteins, the function ofthis C-domain remains unknown.

Arabdopsis AGL8 is a 242 amino acid MADS box protein (see FIG. 7; SEQ IDNO:2; Mandel and Yanofsky, supra, 1995). The AGL8 MADS domain resides atamino acids 2 to 56 of SEQ ID NO:2. The K-domain of AGL8 resides atamino acids 92 to 158 of SEQ ID NO:2.

In wild-type Arabidopsis, AGL8 RNA accumulates in two distinct phases,the first occurring during inflorescence development in the stem andcauline leaves and the second in the later stages of flower development(Mandel and Yanofsky, supra, 1995). In particular, AGL8 RNA is firstdetected in the inflorescence meristem as soon as the plant switchesfrom vegetative to reproductive development. As the inflorescence stemelongates, AGL8 RNA accumulates in the inflorescence meristem and in thestem. Secondly, although AGL8 is not detected in the initial stages (1and 2) of flower development, AGL8 expression resumes at approximatelystage 3 in the center of the floral dome in the region corresponding tothe fourth (carpel) whorl. AGL8 expression is excluded from all otherprimordia and the pedicel. The time of AGL8 expression in the fourthcarpel whorl generally corresponds to the time at which the organidentity genes APETALA3, PISTILLATA AND AGAMOUS begin to be expressed(Yanofsky et al., Nature 346:35-39 (1990); Drews et al., Cell65:991-1002 (1991); Jack et al., Cell 68:683-697 (1992); Goto andMeyerowitz, Genes Devel. 8:1548-1560 (1994)). At later stages, AGL8expression becomes localized to the carpel walls, in the region thatconstitutes the valves of the ovary, and is absent from nearly all othercell types of the carpel. No AGL8 RNA expression is detected in theovules, stigmatic tissues or the septum that divides the ovary. Thus, innature, AGL8 expression during the later stages of floral development isrestricted to the valves of the carpels and to the cells within thestyle.

As used herein, the term “AGL8-like gene product” means a gene productthat has the same or similar function as Arabdopsis AGL8 such that, whenectopically expressed in a vascular plant, normal development isaltered, and the extent of lignification is reduced. Arabdopsis AGL8(SEQ ID NO:2) is an example of an AGL8-like gene product as definedherein. An AGL8-like gene product also can be characterized, in part, byits ability to interact with AGL1 and, additionally, its ability tointeract with AGL5.

An AGL8-like gene product generally is characterized, in part, by havingan amino acid sequence that has at least about 50% amino acid identitywith the amino acid sequence of Arabdopsis AGL8 (SEQ ID NO: 2). AnAGL8-like gene product can have, for example, an amino acid sequencewith greater than about 65% amino acid sequence identity with ArabdopsisAGL8 (SEQ ID NO:2), preferably greater than about 75% amino acididentity with Arabdopsis AGL8 (SEQ ID NO:2), more preferably greaterthan about 85% amino acid identity with Arabdopsis AGL8 (SEQ ID NO:2),and can be a sequence having greater than about 90%, 95% or 97% aminoacid identity with Arabdopsis AGL8 (SEQ ID NO:2). These and other aminoacid identities can be determined, for example, with CLUSTALW using theBLOSUM 62 matrix with default parameters.

Preferably, an AGL8-like gene product is orthologous to the vascularplant species in which it is ectopically expressed. A nucleic acidmolecule encoding Arabdopsis AGL8 (SEQ ID NO:2), for example, can beectopically expressed in an Arabdopsis plant to produce a non-naturallyoccurring Arabdopsis variety characterized by reduced lignification.Similarly, a nucleic acid molecule encoding Eucalyptus AGL8 can beectopically expressed in a Eucalyptus plant to produce a non-naturallyoccurring Eucalyptus variety characterized by reduced lignification.

A nucleic acid molecule encoding an AGL8-like gene product also can beectopically expressed in a heterologous vascular plant to produce anon-naturally occurring vascular plant characterized by reducedlignification. AGAMOUS-like gene products have been widely conservedthroughout the plant kingdom; for example, AGAMOUS has been conserved intomato (TAG1) and maize (ZAG1), indicating that orthologs ofAGAMOUS-like genes are present in most, if not all, angiosperms (Pnueliet al., The Plant Cell 6:163-173 (1994); Schmidt et al., The Plant Cell5:729-737 (1993)). AGL8-like gene products such as AGL8 orthologs alsocan be conserved and can function across species boundaries to reducelignification. Thus, ectopic expression of a nucleic acid moleculeencoding Arabdopsis AGL8 (SEQ ID NO:2) in a heterologous vascular plantsuch as another Brassica and can alter normal development such that theextent of lignification is reduced. Furthermore, a nucleic acid moleculeencoding Arabdopsis AGL8 (SEQ ID NO:2), for example, can be ectopicallyexpressed in more distantly related heterologous vascular plants,including dicotyledonous and monocotyledonous angiosperms andgymnosperms, for example, woody plants, leguminous plants and grassesand, upon ectopic expression, can alter normal development such thatlignification is reduced in the heterologous plant.

As used herein, the term “gene product” encompasses an active segment ofa lignification regulatory gene product such as an AGL8-like geneproduct, which is a polypeptide portion of the gene product that, whenectopically expressed, alters normal development such that lignificationis altered in the same manner as the full-length gene product. An activesegment can be, for example, an amino terminal, internal or carboxyterminal fragment of lignification regulatory factor such as ArabdopsisAGL8 (SEQ ID NO:2) that, when ectopically expressed in a vascular plant,alters normal development such that lignification is reduced orenhanced. An active segment of a MADS-domain containing lignificationregulatory factor can include, for example, the MADS domain and can havethe ability to bind DNA specifically. The skilled artisan will recognizethat a nucleic acid molecule encoding an active segment of alignification regulatory factor such as an AGL8-like gene product can beuseful in producing a vascular plant of the invention characterized byreduced or enhanced lignification.

An active segment of a lignification regulatory factor such as anAGL8-like gene product can be identified using, for example,histochemical tests such as the toluidine blue 0 test, Wiesner's test(specific for cinnamaldehyde groups), or Maule's reaction (specific forsyringyl groups; Strivastava, Tappi 49:173-183 (1966), which isincorporated by reference herein), or cytochemical probes such as thestain with KMnO₄ and Coppick and Fowler's reaction (Czaninski et al.,Biol. Cell. 35: 97-102 (1979); Barceló, supra, 1997, each of which isincorporated by reference herein). Quantitative determination of ligninsalso can be achieved by various direct and indirect methods, includingdirect chemical methods such as the preparation of “Klason lignins” and“thioglycolate lignins” (Effland, Tappi 60:143-144 (1977), which isincorporated by reference herein). Spectrophotometric methods, includingthe acetyl-bromide method consisting of the solubilization of ligninswith acetyl bromide in glacial acetic acid, can be used with lignifiedtissues that do not contain significant amounts of ester-bound cinnamicacids (Johnson et al., Tappi 44:793-798 (1961); Iiyama and Wallis, WoodSci. Technol. 22:271-280 (1988), each of which is incorporated herein byreference). Additional in situ microscopic techniques include UVmicroscopy based on the blue autofluorescence of lignins illuminatedwith UV light (Scott et al., Wood Sci. Technol. 3:73-92 (1969), which isincorporated herein by reference); interference microscopy (Donaldson,N.Z. J. For. Sci. 15:349-360 (1985), which is incorporated herein byreference); and bromination in conjunction with energy-dispersive X-rayanalysis (Saka et al., Tappi 61:73-76 (1978), which is incorporatedherein by reference). If desired, the nature and monomer composition oflignins also can be determined by routine chemical and physical methods(Barteló, supra, 1997).

A vascular plant such as Arabdopsis can be transformed with a nucleicacid molecule under control of a constitutive regulatory element such asa tandem CaMV 35S promoter. Microscopic analysis of toluidine bluesections from transgenic and wild type vascular plants reveals whether aplant ectopically expressing a particular polypeptide portion ischaracterized by reduced lignification. For analysis of a large numberof polypeptide portions of a lignification regulatory gene product,nucleic acid molecules encoding the polypeptide portions can be assayedin pools, and active pools subsequently subdivided to identify theactive nucleic acid molecule.

In one embodiment, the invention provides a non-naturally occurringplant that is characterized by reduced lignification due to ectopicexpression of a nucleic acid molecule encoding an AGL8-like gene producthaving substantially the amino acid sequence of an AGL8 ortholog. Asused herein, the term “AGL8 ortholog” means an ortholog of ArabdopsisAGL8 (SEQ ID NO:2) and refers to an AGL8-like gene product that, in aparticular plant variety, has the highest percentage homology at theamino acid level to Arabdopsis AGL8 (SEQ ID NO:2). An AGL8 ortholog canbe, for example, a Eucalyptus AGL8 ortholog or an alfalfa AGL8 ortholog.An AGL8 ortholog from the long-day plant Sinapis alba, designated SaMADSB, has been described (Menzel et al., Plant J. 9:399-408 (1996), whichis incorporated herein by reference). Similarly, the sequence of thetomato AGL8 ortholog is available as EST244966 under accession numberAI486645. Novel AGL8 ortholog cDNAs can be isolated from additionalplant species using a nucleotide sequence as a probe and methods wellknown in the art of molecular biology (Glick and Thompson (eds.),Methods in Plant Molecular Biology and Biotechnology, Boca Raton, Fla.:CRC Press (1993); Sambrook et al. (eds.), Molecular Cloning: ALaboratory Manual (Second Edition), Plainview, N.Y.: Cold Spring HarborLaboratory Press (1989), each of which is incorporated herein byreference).

As used herein, the term “substantially the amino acid sequence,” whenused in reference to an AGL8 ortholog or an ortholog of anotherlignification regulatory factor, is intended to mean a polypeptide orpolypeptide segment having an identical amino acid sequence, or apolypeptide or polypeptide segment having a similar, non-identicalsequence that is considered by those skilled in the art to be afunctionally equivalent amino acid sequence. For example, an AGL8-likegene product having substantially the amino acid sequence of ArabdopsisAGL8 can have an amino acid sequence identical to the sequence ofArabdopsis AGL8 (SEQ ID NO:2) shown in FIG. 7, or a similar,non-identical sequence that is functionally equivalent. In particular,an amino acid sequence that is “substantially the amino acid sequence”of a lignification regulatory factor such as AGL8 can have one or moremodifications such as amino acid additions, deletions or substitutionsrelative to the Arabdopsis amino acid sequence shown, provided that,when ectopically expressed in the vascular plant, the modifiedpolypeptide retains substantially the ability to alter normaldevelopment such that lignification is reduced in the case of AGL8, orenhanced in the case of AGL1, AGL5 or R-like bHLH. Comparison ofsequences for substantial similarity can be performed between twosequences of any length and usually is performed with sequences betweenabout 6 and 1200 residues, preferably between about 10 and 100 residuesand more preferably between about 25 and 35 residues. Such comparisonsfor substantial similarity are performed using methodology routine inthe art.

It is understood that minor modifications of primary amino acid sequencecan result in a lignification regulatory factor, such as an AGL8-likegene product, that has substantially equivalent or enhanced function ascompared to the ortholog from which it was derived. Further, variousmolecules can be attached to an ortholog or active segment thereof, forexample, other polypeptides, antigenic or other peptide tags,carbohydrates, lipids, or chemical moieties. For example, a heterologousactivation domain can be fused to the full-length sequence, orsubstituted for the naturally occurring activation of one of thedisclosed lignification regulatory factors disclosed herein. Suchmodifications are included within the term “ortholog” as defined herein.

One or more point mutations can be introduced into a nucleic acidmolecule encoding a lignification regulatory factor to yield a modifiednucleic acid molecule using, for example, site-directed mutagenesis (seeWu (Ed.), Meth. In Enzymol. Vol. 217, San Diego: Academic Press (1993);Higuchi, “Recombinant PCR” in Innis et al. (Ed.), PCR Protocols, SanDiego: Academic Press, Inc. (1990), each of which is incorporated hereinby reference). Such mutagenesis can be used to introduce a specific,desired amino acid insertion, deletion or substitution; alternatively, anucleic acid sequence can be synthesized having random nucleotides atone or more predetermined positions to generate random amino acidsubstitutions. Scanning mutagenesis also can be useful in generating amodified nucleic acid molecule encoding substantially the amino acidsequence of an ortholog such as an AGL8 ortholog. Modified nucleic acidmolecules can be routinely assayed for the ability to reducelignification using, for example, microscopic analysis of toluidine bluestained sections or another method described hereinabove.

The methods of the invention can be used to reduce or, as set forthbelow, enhance lignification in one of a variety of vascular plantspecies, including a variety of monocotyledonous and dicotyledonousangiosperms and gymnosperms. As used herein, the term “vascular plant”means a higher plant capable of producing lignins, such as an angiospermor gymnosperm. An angiosperm is a seed-bearing plant whose seeds areborne in a mature ovary (fruit) and are divided into two broad classesbased on the number of cotyledons or seed leaves that generally store orabsorb food. Thus, the invention can be practiced, for example, with amonocotyledonous or dicotyledonous angiosperm, or with a gymnosperm,which is a seed-bearing plant with seeds not enclosed in an ovary.

In one embodiment, the invention provides a method of reducinglignification in a woody plant. Woody plants, including conifer andhardwood trees such as, for example, Eucalyptus, cottonwood, alder,Douglas fir, Hemlock, pine and spruce, can be modified as disclosedherein to produce a tree characterized by reduced lignification. Theskilled person understands that the invention can be practiced withthese or other woody plants or trees, especially trees useful forproducing pulp or paper (Whetten and Sederoff, Forest Ecology andManagement 43:301-316 (1991), which is incorporated herein byreference).

In another embodiment, the invention provides a method of reducinglignification in a leguminous plant. A leguminous plant can produce aforage legume, such as alfalfa, lucerne, birdsfoot trefoil, clover,Stylosanthes species, Lotononis bainessii or sainfoin (Buxton andRedfearn, J. Nutr. 127:814S-818S (1997); Dixon et al., Gene 179:61-71(1996), each of which is incorporated herein by reference). The skilledartisan will recognize that these or other leguminous plants can bemodified as disclosed herein to produce a non-naturally occurring plantvariety characterized by reduced lignification.

In another embodiment, the invention provides a method of reducinglignification in a forage grass, such forage grasses useful as they aremore easily digestible by cattle. The methods of the invention can beused, for example, to reduce lignification in a forage grass such asbahiagrass, bermudagrass, dallisgrass, pangolagrass, big bluestem,indiangrass, switchgrass, smooth bromegrass, orchardgrass, timothy,Kentucky bluegrass or tall fescue.

The term “exogenous,” as used herein in reference to a nucleic acidmolecule and a transgenic vascular plant, means a nucleic acid moleculeoriginating from outside the vascular plant. An exogenous nucleic acidmolecule can be, for example, a nucleic acid molecule encoding anAGL8-like gene product or an exogenous regulatory element such as aconstitutive or tissue-selective regulatory element. An exogenousnucleic acid molecule can have a naturally occurring or non-naturallyoccurring nucleotide sequence and can be a heterologous nucleic acidmolecule derived from a different species than the plant into which thenucleic acid molecule is introduced or can be a nucleic acid moleculederived from the same plant species as the plant into which it isintroduced.

The term “operatively linked,” as used in reference to a regulatoryelement and a nucleic acid molecule, means that the regulatory elementconfers regulated expression upon the operatively linked nucleic acidmolecule. Thus, the term “operatively linked,” as used in reference toan exogenous regulatory element such as a constitutive regulatoryelement and a nucleic acid molecule encoding a lignification regulatoryfactor such as an AGL8-like gene product, means that the constitutiveregulatory element is linked to the nucleic acid molecule encoding anAGL8-like gene product such that a constitutive expression pattern isconferred upon the nucleic acid molecule encoding the AGL8-like geneproduct. It is recognized that a regulatory element and a nucleic acidmolecule that are operatively linked have, at a minimum, all elementsessential for transcription, including, for example, a TATA box.

As used herein, the term “constitutive regulatory element” means aregulatory element that confers a level of expression upon anoperatively linked nucleic molecule that is relatively independent ofthe cell or tissue type in which the constitutive regulatory element isexpressed. A constitutive regulatory element that is expressed in avascular plant generally is widely expressed in a large number of celland tissue types.

A variety of constitutive regulatory elements useful for ectopicexpression in a transgenic vascular plant are well known in the art. Thecauliflower mosaic virus 35S (CaMV 35S) promoter, for example, is awell-characterized constitutive regulatory element that produces a highlevel of expression in all plant tissues (Odell et al., Nature313:810-812 (1985)). The CaMV 35S promoter can be particularly usefuldue to its activity in numerous diverse plant species (Benfey and Chua,Science 250:959-966 (1990); Futterer et al., Physiol. Plant 79:154(1990); Odell et al., supra, 1985). A tandem 35S promoter, in which theintrinsic promoter element has been duplicated, confers higherexpression levels in comparison to the unmodified 35S promoter (Kay etal., Science 236:1299 (1987)). Other constitutive regulatory elementsuseful for ectopically expressing a nucleic acid molecule encoding anAGL8-like gene product in a transgenic vascular plant of the inventioninclude, for example, the cauliflower mosaic virus 19S promoter; theFigwort mosaic virus promoter; and the nopaline synthase (nos) genepromoter (Singer et al., Plant Mol. Biol. 14:433 (1990); An, PlantPhysiol. 81:86 (1986)).

Additional constitutive regulatory elements including those forefficient ectopic expression in monocots also are known in the art, forexample, the pEmu promoter and promoters based on the rice Actin-1 5′region (Last et al., Theor. Appl. Genet. 81:581 (1991); Mcelroy et al.,Mol. Gen. Genet. 231:150 (1991); Mcelroy et al., Plant Cell 2:163(1990)). Chimeric regulatory elements, which combine elements fromdifferent genes, also can be useful for ectopically expressing a nucleicacid molecule encoding an AGL8-like gene product (Comai et al., PlantMol. Biol. 15:373 (1990)). One skilled in the art understands that aparticular constitutive regulatory element is chosen based, in part, onthe plant species in which a nucleic acid molecule encoding an AGL8-likegene product is to be ectopically expressed and on the desired level ofexpression.

An exogenous regulatory element useful in a transgenic vascular plant ofthe invention also can be an inducible regulatory element, which is aregulatory element that confers conditional expression upon anoperatively linked nucleic acid molecule, where expression of theoperatively linked nucleic acid molecule is increased in the presence ofa particular inducing agent or stimulus as compared to expression of thenucleic acid molecule in the absence of the inducing agent or stimulus.Particularly useful inducible regulatory elements includecopper-inducible regulatory elements (Mett et al., Proc. Natl. Acad.Sci. USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717 (1988));tetracycline and chlor-tetracycline-inducible regulatory elements (Gatzet al., Plant J. 2:397-404 (1992); Röder et al., Mol. Gen. Genet.243:32-38 (1994); Gatz, Meth. Cell Biol. 50:411-424 (1995)); ecdysoneinducible regulatory elements (Christopherson et al., Proc. Natl. Acad.Sci. USA 89:6314-6318 (1992); Kreutzweiser et al., Ecotoxicol. Environ.Safety 28:14-24 (1994)); heat shock inducible regulatory elements(Takahashi et al., Plant Physiol. 99:383-390 (1992); Yabe et al., PlantCell Physiol. 35:1207-1219 (1994); Ueda et al., Mol. Gen. Genet.250:533-539 (1996)); and lac operon elements, which are used incombination with a constitutively expressed lac repressor to confer, forexample, IPTG-inducible expression (Wilde et al., EMBO J. 11:1251-1259(1992)).

An inducible regulatory element useful in the transgenic vascular plantsof the invention also can be, for example, a nitrate-inducible promoterderived from the spinach nitrite reductase gene (Back et al., Plant Mol.Biol. 17:9 (1991)) or a light-inducible promoter, such as thatassociated with the small subunit of RuBP carboxylase or the LHCP genefamilies (Feinbaum et al., Mol. Gen. Genet. 226:449 (1991); Lam andChua, Science 248:471 (1990)). Additional inducible regulatory elementsinclude salicylic acid inducible regulatory elements (Uknes et al.,Plant Cell 5:159-169 (1993); Bi et al., Plant J. 8:235-245 (1995));plant hormone-inducible regulatory elements (Yamaguchi-Shinozaki et al.,Plant Mol. Biol. 15:905 (1990); Kares et al., Plant Mol. Biol. 15:225(1990)); and human hormone-inducible regulatory elements such as thehuman glucocorticoid response element (Schena et al., Proc. Natl. Acad.Sci. USA 88:10421 (1991)).

Tissue-selective regulatory elements also can be useful in theinvention. Such a tissue-selective regulatory element, which can be usedto ectopically express a nucleic acid molecule in a single tissue or ina limited number of tissues, can be, for example, a xylem-selectiveregulatory element, a tracheid-selective regulatory element or afiber-selective regulatory element. Such tissue-selective regulatoryelements are known in the art or can be isolated using routinemethodology (Glick and Thompson, supra, 1993).

A dehiscence zone-selective regulatory element also can be atissue-selective regulatory element useful in the invention. Adehiscence zone-selective regulatory element can be derived from AGL1 orAGL5 or a gene that is an ortholog of Arabdopsis AGL1 or AGL5 and isselectively expressed in the valve margin or dehiscence zone of avascular plant. Dehiscence zone-selective regulatory elements also canbe derived from a variety of other genes that are selectively expressedin the valve margin or dehiscence zone of a vascular plant. For example,the rapeseed gene RDPG1 is selectively expressed in the dehiscence zone(Petersen et al., Plant Mol. Biol. 31:517-527 (1996), which isincorporated herein by reference). Thus, the RDPG1 promoter or an activefragment thereof can be a dehiscence zone-selective regulatory elementas defined herein. Additional genes such as the rapeseed gene SAC51 alsoare known to be selectively expressed in the dehiscence zone; the SAC51promoter or an active fragment thereof also can be a dehiscencezone-selective regulatory element of the invention (Coupe et al., PlantMol. Biol. 23:1223-1232 (1993), which is incorporated herein byreference). Further, genes selectively expressed in the dehiscence zoneinclude the R-like bHLH gene that confers selective GUS expression inthe Arabdopsis transposant line GT140 (Sundaresan et al., Genes Devel.9:1797-1810 (1995), which is incorporated herein by reference).

Transgenic vascular plants characterized by reduced lignification alsoare provided by the invention. Such transgenic plants contain anectopically expressed nucleic acid molecule including a lignifiedtissue-selective regulatory element operatively linked to a nucleic acidmolecule encoding an AGL8-like gene product. The AGL8-like gene productcan have, for example, substantially the amino acid sequence of an AGL8ortholog and can be, for example, Arabdopsis AGL8 (SEQ ID NO: 2). In atransgenic vascular plant of the invention characterized by reducedlignification, the lignified tissue-selective regulatory element can be,for example, a fiber-selective regulatory element, xylem-selectiveregulatory element or tracheid selective regulatory element.

Further provided herein is a tissue derived from a transgenic vascularplant containing an ectopically expressed nucleic acid molecule thatcontains a lignified tissue-selective regulatory element operativelylinked to a nucleic acid molecule encoding an AGL8-like gene product.

As used herein, the term “transgenic” refers to a vascular plant thatcontains an exogenous nucleic acid molecule, which can be derived fromthe same plant species or a heterologous plant species.

As used herein, the term “lignified tissue-selective regulatory element”refers to a nucleotide sequence that, when operatively linked to anucleic acid molecule, confers selective expression upon the operativelylinked nucleic acid molecule in a limited number of vascular planttissues, including one or more lignified tissues such as fiber, xylem ortracheid tissue. One skilled in the art understands that a lignifiedtissue-selective regulatory element can confer specific expressionexclusively in cells that normally are lignified in a wild type vascularplant or can confer selective expression in a limited number of plantcell types including lignified cells.

Lignified tissue-selective regulatory elements are known in the art, forexample, a useful xylem-selective regulatory element can be aphenylalanine ammonia-lyase (PAL) promoter sequence. Poplar PAL1 or PAL2promoter sequences, for example, can be used to direct xylem-selectiveexpression in heterologous transgenic plants, for example, in developingprimary xylem of leaves, stems and other organs and in secondary xylemof stems (Gray-Mitsumune et al., Plant Mol. Biol. 39:657-659 (1999);accession number AF038863 and AF038864).

It should be recognized that a non-naturally occurring vascular plant ofthe invention, which contains an ectopically expressed nucleic acidmolecule encoding an AGL8-like gene product, also can contain one ormore additional modifications, including naturally and non-naturallyoccurring modifications, that can modulate the reduction inlignification or provide other advantageous properties. Such vascularplants are encompassed within the invention.

The methods of the invention entail ectopically expressing a nucleicacid molecule encoding a lignification regulatory factor to alter thenatural lignification process. In one embodiment, the methods includeintroducing an ectopically expressible nucleic acid molecule encoding anAGL8-like gene product into the vascular plant, whereby lignification isreduced due to ectopic expression of the nucleic acid molecule.

As discussed above, the term “ectopically” refers to expression of anucleic acid molecule encoding a lignification regulatory factor in acell type other than a cell type in which the nucleic acid molecule isnormally expressed, at a time other than a time at which the nucleicacid molecule is normally expressed or at an expression level other thanthe level at which the nucleic acid normally is expressed. In wild typeArabidopsis, for example, AGL8 expression is normally restricted duringthe later stages of floral development to the carpel valves. In themethods of the invention, particularly useful ectopic expression of anucleic acid molecule encoding an AGL8-like gene product involvesexpression in cell types that in wild type plants can be lignified, forexample, within the stem or xylem, leaves, or fruit, such as adjacent tothe dehiscence zone.

Actual ectopic expression of lignification regulatory factor isdependent on various factors. The ectopic expression can be widespreadexpression throughout most or all plant tissues or can be expressionrestricted to a small number of plant tissues, and can be achieved by avariety of routine techniques. Mutagenesis, including seed or pollenmutagenesis, can be used to generate a non-naturally occurring vascularplant, in which a nucleic acid molecule encoding a lignificationregulatory factor, for example, an AGL8-like gene product is ectopicallyexpressed. Ethylmethane sulfonate (EMS) mutagenesis, transposon mediatedmutagenesis or T-DNA mediated mutagenesis also can be useful inectopically expressing a lignification regulatory factor to produce avascular plant characterized by reduced or enhanced lignification (see,generally, Glick and Thompson, supra, 1993). While not wishing to bebound by any particular mechanism, ectopic expression in a mutagenizedplant can result from inactivation of one or more negative regulators ofa lignification regulatory factor, for example, ectopic expression ofAGL8 can result from the combined inactivation of AGL1 and AGL5.

Ectopic expression of a lignification regulatory factor such as anAGL8-like gene product also can be achieved by expression of a nucleicacid encoding the regulatory factor from a heterologous regulatoryelement or from a modified variant of its own promoter. Heterologousregulatory elements include constitutive regulatory elements, whichresult in expression of the lignification regulatory factor in most orall plant cell types, and tissue-selective regulatory elements, whichproduce selective expression of the lignification regulatory factor in alimited number of cell types.

Ectopic expression of a nucleic acid molecule encoding a lignificationregulatory factor such as an AGL8-like gene product can be achievedusing an endogenous or exogenous nucleic acid molecule encoding theregulatory factor. A recombinant exogenous nucleic acid molecule cancontain a heterologous regulatory element that is operatively linked toa nucleic acid sequence encoding the lignification regulatory factor.Methods for producing the desired recombinant nucleic acid moleculeunder control of a heterologous regulatory element and for producing anon-naturally occurring vascular plant of the invention are well knownin the art (see, generally, Sambrook et al., supra, 1989; Glick andThompson, supra, 1993).

An exogenous nucleic acid molecule can be introduced into a vascularplant for ectopic expression using a variety of transformationmethodologies including Agrobacterium-mediated transformation and directgene transfer methods such as electroporation andmicroprojectile-mediated transformation (see, generally, Wang et al.(eds), Transformation of Plants and Soil Microorganisms, Cambridge, UK:University Press (1995), which is incorporated herein by reference).Transformation methods based upon the soil bacterium Agrobacteriumtumefaciens are particularly useful for introducing an exogenous nucleicacid molecule into a vascular plant. The wild type form of Agrobacteriumcontains a Ti (tumor-inducing) plasmid that directs production oftumorigenic crown gall growth on host plants. Transfer of thetumor-inducing T-DNA region of the Ti plasmid to a plant genome requiresthe Ti plasmid-encoded virulence genes as well as T-DNA borders, whichare a set of direct DNA repeats that delineate the region to betransferred. An Agrobacterium-based vector is a modified form of a Tiplasmid, in which the tumor inducing functions are replaced by thenucleic acid sequence of interest to be introduced into the plant host.

Agrobacterium-mediated transformation generally employs cointegratevectors or, preferably, binary vector systems, in which the componentsof the Ti plasmid are divided between a helper vector, which residespermanently in the Agrobacterium host and carries the virulence genes,and a shuttle vector, which contains the gene of interest bounded byT-DNA sequences. A variety of binary vectors are well known in the artand are commercially available, for example, from Clontech (Palo Alto,Calif.). Methods of coculturing Agrobacterium with cultured plant cellsor wounded tissue such as leaf tissue, root explants, hypocotyledons,stem pieces or tubers, for example, also are well known in the art(Glick and Thompson, supra, 1993). Wounded cells within the plant tissuethat have been infected by Agrobacterium can develop organs de novo whencultured under the appropriate conditions; the resulting transgenicshoots eventually give rise to transgenic plants that ectopicallyexpress a nucleic acid molecule encoding an AGL8-like gene product.Agrobacterium also can be used for transformation of whole plants asdescribed in Bechtold et al., C.R. Acad. Sci. Paris, Life Sci.316:1194-1199 (1993), which is incorporated herein by reference).Agrobacterium-mediated transformation is useful for producing a varietyof transgenic vascular plants (Wang et al., supra, 1995) including atleast one species of Eucalyptus and forage legumes such as alfalfa(lucerne); birdsfoot trefoil, white clover, Stylosanthes, Lotononisbainessii and sainfoin.

Microprojectile-mediated transformation also can be used to produce atransgenic vascular plant that ectopically expresses a lignificationregulatory factor. This method, first described by Klein et al. (Nature327:70-73 (1987), which is incorporated herein by reference), relies onmicroprojectiles such as gold or tungsten that are coated with thedesired nucleic acid molecule by precipitation with calcium chloride,spermidine or PEG. The microprojectile particles are accelerated at highspeed into an angiosperm tissue using a device such as the BIOLISTICPD-1000 (Biorad; Hercules Calif.).

Microprojectile-mediated delivery or “particle bombardment” isespecially useful to transform plants that are difficult to transform orregenerate using other methods. Microprojectile-mediated transformationhas been used, for example, to generate a variety of transgenic vascularplant species, including cotton, tobacco, corn, hybrid poplar and papaya(see Glick and Thompson, supra, 1993) as well as cereal crops such aswheat, oat, barley, sorghum and rice (Duan et al., Nature Biotech.14:494-498 (1996); Shimamoto, Curr. Opin. Biotech. 5:158-162 (1994),each of which is incorporated herein by reference). In view of theabove, the skilled artisan will recognize that Agrobacterium-mediated ormicroprojectile-mediated transformation, as disclosed herein, or othermethods known in the art can be used to introduce a nucleic acidmolecule encoding a lignification regulatory factor such as an AGL8-likegene product into a vascular plant for ectopic expression.

The invention also provides a method of reducing lignification in avascular plant by suppressing both AGL1 and AGL5 expression in thevascular plant, whereby lignification is reduced.

As disclosed herein, loss-of-function mutations in the AGL1 and AGL5genes were produced by a combination of homologous recombination anddisruptive T-DNA insertion (see Example II). Neither AGL1 nor AGL5 RNAwas expressed in the resulting agl1 agl5 double mutant, and toluidineblue staining and microscopy revealed that the patches of cells whichnormally are lignified in wild type plants are absent in the agl1 agl5mutant. These results indicate that AGL1 or AGL5 gene expression isrequired for normal lignification and that suppression AGL1 expressioncombined with suppression of AGL5 expression in the vascular plant canreduce lignification, allowing plant varieties with improvedcharacteristics to be developed.

The Arabdopsis AGL1 and AGL5 genes encode MADS box proteins with 85%identity at the amino acid level (see Tables 1 and 2). The AGL1 and AGL5RNA expression patterns also are strikingly similar. In particular, bothRNAs are specifically expressed in flowers, where they accumulate indeveloping carpels. In particular, strong expression of these genes isobserved in the outer replum along the valve/replum boundary (Ma et al.,supra, 1991; Savidge et al., The Plant Cell 7:721-723 (1995); Flanaganet al., The Plant Journal 10:343-353 (1996), each of which isincorporated herein by reference).

TABLE 1 Amino acid identity in the MADS domain and K-domain of AGAMOUS,AGL1 and AGL5 AGAMOUS AGL1 AGL5 MADS K MADS K MADS K AGAMOUS — — 95% 68% 95% 62% AGL1 — — — — 100% 92% AGL5 — — — — — —

TABLE 2 Amino acid identity in the I-domain and C-domain of AGAMOUS,AGL1 and AGL5 AGAMOUS AGL1 AGL5 I C I C I C AGAMOUS — — — — — — AGL1 71%39% — — — — AGL5 65% 37% 95% 72% — —

As used herein, the term “AGL1” refers to Arabdopsis AGL1 (SEQ ID NO:4)or an ortholog of Arabdopsis AGL1 (SEQ ID NO:4). An AGL1 ortholog is aMADS box gene product characterized, in part, by positively regulatingthe process of lignification and, in part, by having homology to theamino acid sequence of Arabdopsis AGL1 (SEQ ID NO:4). AGL1 or an AGL1ortholog can function, in part, by forming a complex with an AGL8-likegene product. An AGL1 ortholog generally has an amino acid sequencehaving at least about 63% amino acid identity with Arabdopsis AGL1 (SEQID NO:4) and includes polypeptides having greater than about 70%, 75%,85% or 95% amino acid identity with Arabdopsis AGL1 (SEQ ID NO:4). Giventhe close relatedness of the AGL1 and AGL5 gene products, one skilled inthe art will recognize that an AGL1 ortholog can be distinguished froman AGL5 ortholog by being more closely related to Arabidopsis AGL1 (SEQID NO:4) than to Arabdopsis AGL5 (SEQ ID NO:6). An AGL1 ortholog canfunction in wild type plants, like Arabdopsis AGL1, to limit the domainof AGL8-like gene product expression.

As used herein, the term “AGL5” refers to Arabdopsis AGL5 (SEQ ID NO:6)or to an ortholog of Arabdopsis AGL5 (SEQ ID NO:6). An AGL5 ortholog isa MADS box gene product characterized, in part, by positively regulatingthe process of lignification and, in part, by having homology to theamino acid sequence of Arabdopsis AGL5 (SEQ ID NO:6). AGL5 or an AGL5ortholog can function, in part, by forming a complex with an AGL8-likegene product. An AGL5 ortholog generally has an amino acid sequencehaving at least about 60% amino acid identity with Arabdopsis AGL5 (SEQID NO:6) and includes polypeptides having greater than about 65%, 70%,75%, 85% or 95% amino acid identity with Arabdopsis AGL5 (SEQ ID NO:6).Given the close relatedness of the AGL1 and AGL5 gene products, oneskilled in the art will recognize that an AGL5 ortholog can bedistinguished from an AGL1 ortholog by being more closely related toArabdopsis AGL5 (SEQ ID NO:6) than to Arabdopsis AGL1 (SEQ ID NO:4). AnAGL5 ortholog can function in wild type plants, like Arabdopsis AGL5, tolimit the domain of AGL8-like gene product expression.

The invention further provides a method of reducing lignification in avascular plant by suppressing R-like bHLH expression in said vascularplant, whereby lignification is reduced. In addition, the inventionprovides a non-naturally occurring vascular plant characterized byreduced lignification, in which R-like bHLH expression in suppressed,whereby lignification is reduced. In one embodiment, the non-naturallyoccurring vascular plant does not have suppressed R-like bHLH expressiondue to ectopic expression of AGL8 or due to suppressed AGL1 and AGL5expression.

As used herein, the term “R-like bHLH gene product” means a gene productthat has the same or similar function as Arabdopsis R-like bHLH suchthat, when ectopically expressed in a vascular plant, normal developmentis altered, and the extent of lignification is enhanced. Thus, a R-likebHLH gene product is characterized, in part, in that it is a positiveregulator of lignification.

A R-like bHLH gene product generally is characterized, in part, byhaving an amino acid sequence that has at least about 50% amino acididentity with the amino acid sequence of Arabdopsis R-like bHLH (SEQ IDNO: 25). A R-like bHLH gene product can have, for example, an amino acidsequence with greater than about 65% amino acid sequence identity withArabdopsis R-like bHLH (SEQ ID NO:25), preferably greater than about 75%amino acid identity with SEQ ID NO:25, more preferably greater thanabout 85% amino acid identity with SEQ ID NO:25, and can be a sequencehaving greater than about 90%, 95% or 97% amino acid identity with SEQID NO:25.

The term “suppressed,” as used herein in reference to AGL1 expression,means that the amount of functional AGL1 protein is reduced in a plantin comparison with the amount of functional AGL1 protein in thecorresponding wild type plant. Similarly, when used in reference to AGL5expression, the term suppressed means that the amount of functional AGL5protein is reduced in a plant in comparison with the amount offunctional AGL5 protein in the corresponding wild type plant. When usedin reference to R-like bHLH expression, the term “suppressed” means thatthe amount of functional R-like bHLH protein is reduced in a plant incomparison with the amount of functional R-like bHLH protein in thecorresponding wild type plant. Thus, the term “suppressed,” as usedherein, encompasses the absence of AGL1, AGL5 or R-like bHLH protein ina plant, as well as protein expression that is present but reduced ascompared to the level of expression of the corresponding protein in awild type plant. Furthermore, the term “suppressed” refers to AGL1, AGL5or R-like bHLH protein expression that is reduced throughout the entiredomain of AGL1, AGL5 or R-like bHLH expression, or to expression that isreduced in some part of the AGL1, AGL5 or R-like bHLH expression domain,provided that the resulting plant is characterized by reducedlignification.

As used herein, the term “suppressed” also encompasses an amount ofAGL1, AGL5 or R-like bHLH protein that is equivalent to wild type AGL1,AGL5 or R-like bHLH expression, but where the AGL1, AGL5 or R-like bHLHprotein has a reduced level of activity. For example, AGL1 and AGL5 eachcontain a conserved MADS domain; point mutations or gross deletionswithin the MADS domain that reduce the DNA-binding activity of AGL1 orAGL5 can reduce or destroy the activity of AGL1 or AGL5 and, therefore,“suppress” AGL1 or AGL5 expression as defined herein.

A variety of methodologies can be used to suppress AGL1, AGL5 or R-likebHLH expression in a vascular plant. Suppression can be achieved bydirectly modifying the AGL1, AGL5 or R-like bHLH genomic locus, forexample, by modifying a regulatory sequence such that transcription ortranslation from the AGL1, AGL5 or R-like bHLH locus is reduced, or bymodifying an AGL1, AGL5 or R-like bHLH coding sequence such thatnon-functional protein is produced. Suppression of AGL1, AGL5 or R-likebHLH expression in a vascular plant also can be achieved indirectly, forexample, by modifying the expression or activity of a protein thatregulates AGL1, AGL5 or R-like bHLH expression. Methodologies foreffecting suppression of protein expression in a plant include, forexample, homologous recombination, chemical and transposon-mediatedmutagenesis, cosuppression and antisense-based techniques and dominantnegative methodologies.

Homologous recombination can be used to suppress expression in avascular plant as described in Kempin et al., Nature 389:802-803 (1997),which is incorporated herein by reference. Homologous recombination canbe used, for example, to replace the wild type AGL5 genomic sequencewith a construct in which the gene for kanamycin resistance is flankedby at least about 1 kb of AGL5 sequence. The use of homologousrecombination to suppress AGL5 expression is set forth in Example II.

Suppression of AGL1, AGL5 or R-like bHLH expression also can be achievedby producing a loss-of-function mutation using transposon-mediatedinsertional mutagenesis with Ds transposons or Stm transposons (see, forexample, Sundaresan et al., Genes Devel. 9:1797-1810 (1995), which isincorporated herein by reference). Insertion of a transposon into anAGL1, AGL5 or R-like bHLH target gene can be identified, for example, byrestriction mapping, which can identify the presence of an insertion inthe gene promoter or in the coding region, such that expression offunctional gene product is suppressed. Insertion of a transposon alsocan be identified by detecting an absence of the mRNA encoded by thetarget gene or by the detecting the absence of the gene product in valvemargin. Suppression of AGL1, AGL5 or R-like bHLH expression also can beachieved by producing a loss-of-function mutation using T-DNA-mediatedinsertional mutagenesis (see Krysan et al., Proc. Natl. Acad. Sci., USA93:8145-8150 (1996)). The use of T-DNA-mediated insertional mutagenesisto suppress AGL1 expression is disclosed in Example II.

Suppression of AGL1, AGL5 or R-like bHLH expression in a vascular plantalso can be achieved using cosuppression, which is a well knownmethodology that relies on expression of a nucleic acid molecule in thesense orientation to produce coordinate silencing of the introducednucleic acid molecule and the homologous endogenous gene (see, forexample, Flavell, Proc. Natl. Acad. Sci., USA 91:3490-3496 (1994);Kooter and Mol, Current Opin. Biol. 4:166-171 (1993), each of which isincorporated herein by reference). Cosuppression is induced moststrongly by a large number of transgene copies or by overexpression oftransgene RNA and can be enhanced by modification of the transgene suchthat it fails to be translated.

Antisense nucleic acid molecules encoding AGL1, AGL5 or R-like bHLH geneproducts, or fragments thereof, also can be used to suppress expressionof the corresponding gene product in a vascular plant. Antisense nucleicacid molecules reduce mRNA translation or increase mRNA degradation,thereby suppressing gene expression (see, for example, Kooter and Mol,supra, 1993; Pnueli et al., The Plant Cell Vol. 6, 175-186 (1994), whichis incorporated herein by reference).

To suppress both AGL1 and AGL5 expression or R-like bHLH expression, theone or more sense or antisense nucleic acid molecules can be expressedunder control of a strong regulatory element. The constitutive CaMV 35Spromoter (Odell et al., supra, 1985), for example, or other constitutivepromoters as disclosed herein, can be useful in the methods of theinvention. Tissue-selective regulatory elements also can be useful forexpressing one or more sense or antisense nucleic acid molecules inorder to suppress AGL1 and AGL5, or R-like bHLH expression, in avascular plant.

The skilled artisan will recognize that effective suppression ofendogenous AGL1, AGL5 or R-like bHLH gene expression depends upon theone or more introduced nucleic acid molecules having a high percentageof homology with the corresponding endogenous gene loci. Nucleic acidmolecules encoding Arabidopsis AGL1 (SEQ ID NO:3), Arabdopsis AGL5 (SEQID NO:5) and Arabdopsis R-like bHLH (SEQ ID NO:24) are provided herein(see, also, Ma et al., supra, 1991). Nucleic acid molecules encodingArabdopsis AGL1, AGL5 and R-like bHLH can be useful in the methods ofthe invention or for isolating orthologous sequences.

The homology requirement for effective suppression using homologousrecombination, cosuppression or antisense methodology can be determinedempirically. In general, a minimum of about 80-90% nucleic acid sequenceidentity is preferred for effective suppression of AGL1, AGL5 or R-likebHLH expression. Thus, a nucleic acid molecule encoding a gene orthologfrom the family or genus of the plant species into which the nucleicacid molecule is to be introduced is preferred for generating thenon-naturally occurring vascular plants of the invention usinghomologous recombination, cosuppression or antisense technology. Morepreferably, a nucleic acid molecule encoding a gene ortholog from thesame plant species is used for suppression in a vascular plant of theinvention.

Although use of a highly homologous nucleic acid molecule is preferredin the methods of the invention, the nucleic acid molecule to be usedfor homologous recombination, cosuppression or antisense suppressionneed not contain in its entirety the sequence to be suppressed. Thus, asense or antisense nucleic acid molecule encoding only a portion ofArabdopsis AGL1 (SEQ ID NO:3), for example, or a sense or antisensenucleic acid molecule encoding only a portion of Arabdopsis AGL5 (SEQ IDNO:5), or a sense or antisense nucleic acid molecule encoding only aportion of Arabdopsis R-like bHLH (SEQ ID NO:24), can be useful forproducing a non-naturally occurring vascular plant of the invention, inwhich AGL1 and AGL5 expression each are suppressed or in which R-likebHLH expression is suppressed.

A portion of a nucleic acid molecule to be homologously recombined witha genomic locus generally contains at least about 1 kb of sequencehomologous to the targeted gene and preferably contains at least about 2kb, more preferably at least about 3 kb and can contain at least about 5kb of sequence homologous to the targeted gene. For example, a portionof a nucleic acid molecule encoding an AGL1 or AGL5 to be used forcosuppression or antisense suppression generally contains at least about50 base pairs to the full-length of the nucleic acid molecule encodingthe AGL1 or AGL5 ortholog. In contrast to an active segment, as definedherein, a portion of a nucleic acid molecule to be used for homologousrecombination, cosuppression or antisense suppression need not encode afunctional part of a gene product.

A dominant negative construct also can be used to suppress AGL1, AGL5 orR-like bHLH expression in a vascular plant. A dominant negativeconstruct useful in the invention generally contains a portion of thecomplete AGL1, AGL5 or R-like bHLH coding sequence sufficient, forexample, for DNA-binding or for a protein-protein interaction such as ahomodimeric or heterodimeric protein-protein interaction but lacking thetranscriptional activity of the wild type protein. For example, acarboxy-terminal deletion mutant of AGAMOUS was used as a dominantnegative construct to suppress expression of the MADS box gene AGAMOUS(Mizukami et al., Plant Cell 8:831-844 (1996), which is incorporated byreference herein). One skilled in the art understands that, similarly, adominant negative AGL1, AGL5 or R-like bHLH construct can be used tosuppress AGL1, AGL5 or R-like bHLH expression in a plant. A useful AGL1or AGL5 dominant negative construct can be a deletion mutant encoding,for example, the MADS box domain alone (“M”), the MADS box domain and“intervening” region (“MI”); the MADS box, “intervening” and “K” domains(“MIK”); or the “intervening,” “K” and carboxy-terminal domains (“IKC”).

The methods of the invention for reducing lignification by suppressingboth AGL1 and AGL5 expression encompass introducing a loss-of-functionmutation at the AGL1 locus and a loss-of-function mutation at the AGL5locus. Loss-of-function mutations encompass point mutations, includingsubstitutions, deletions and insertions, as well as gross modificationsof an AGL1 and AGL5 locus and can be located in coding or non-codingsequences. One skilled in the art understands that any suchloss-of-function mutation at the AGL1 locus can be combined with anysuch mutation at the AGL5 locus to generate an agl1 agl5 double mutantof the invention. Production of an exemplary agl1 agl5 double mutant inthe Brassica plant Arabdopsis is disclosed herein in Example II.

AGL1 and AGL5 are closely related genes that have diverged relativelyrecently. While not wishing to be bound by the following, some plantscan contain only AGL1 or only AGL5, or can contain a single ancestralgene related to AGL1 and AGL5. In such plants, reduced lignification canbe produced by suppressing only expression of AGL1, or expression onlyof AGL5, or expression of a single ancestral gene related to AGL1 andAGL5. Thus, the present invention provides a non-naturally occurringvascular plant characterized by reduced lignification, in which AGL1expression is suppressed, for example, an agl1 single mutant. Thepresent invention also provides a non-naturally occurring vascular plantcharacterized by reduced lignification, in which AGL5 expression issuppressed, for example, an agl5 single mutant.

The present invention further provides a tissue derived from anon-naturally occurring vascular plant of the invention characterized byreduced lignification. In one embodiment, the invention provides atissue derived from a non-naturally occurring vascular plant that ischaracterized by reduced lignification due to ectopic expression of anucleic acid molecule encoding an AGL8-like gene product. In anotherembodiment, the invention provides a tissue derived from a non-naturallyoccurring vascular plant characterized by reduced lignification, inwhich AGL1 expression and AGL5 expression both are suppressed.

As used herein, the term “tissue” means an aggregate of plant cells andintercellular material organized into a structural and functional unit.A particular useful tissue of the invention is a tissue that can bevegetatively or non-vegetatively propagated such that the vascular plantfrom which the tissue was derived is reproduced. A tissue of theinvention can be, for example, a stem, leaf, fruit or part thereof.

Based on identification of AGL1, AGL5, AGL8 and R-like bHLH asregulators of lignification, the invention also provides methods ofenhancing lignification in a vascular plant. As disclosed herein,transgenic plants constitutively expressing AGL1 or AGL5 exhibitenhanced lignification, as indicated by ectopic lignification of thevalve mesophyll layers revealed by staining with toluidine blue orphloroglucinol (see Example IV). As further disclosed herein, an agl8mutant, in which AGL8 expression is suppressed, is characterized byenhanced lignification in that all the internal mesophyll cell layersare lignified rather than the single lignified enb layer in wild typefruit (see Example III). Based on the above, the invention providesmethods of enhancing lignification by altering the natural levels andexpression patterns of transcription factors that regulate thelignification pathway.

Non-naturally occurring plant varieties exhibiting enhancedlignification can be desirable, for example, for improved mechanicalproperties such as greater wind or water resistance, or increasedresistance to plant pathogens. Cereal plants, for example, can bemodified as disclosed herein to produce a non-naturally occurringvariety characterized by enhanced lignification. High levels of ligninin wood increase the intrinsic heat content for direct utilization asfuel. Thus, enhanced lignification can be used to produce woody plantvarieties with improved properties.

Based on the above, the invention provides methods of enhancinglignification in a vascular plant by ectopically expressing a nucleicacid molecule encoding an AGL1/5-like gene product, wherebylignification is enhanced due to ectopic expression of the nucleic acidmolecule. In a method of the invention for enhancing lignification, theAGL1/5-like gene product can have substantially the amino acid sequenceof an AGL1 ortholog and can be, for example, Arabdopsis AGL1 (SEQ IDNO:4). An AGL1/5-like gene product also can have, for example,substantially the amino acid sequence of an AGL5 ortholog and can be,for example, Arabdopsis AGL5 (SEQ ID NO: 6). The methods of theinvention can be particularly valuable for enhancing lignification inwoody plants or trees that are produced for direct utilization as fuel.

In one embodiment, the invention provides a method of enhancinglignification in a vascular plant by introducing an exogenous nucleicacid molecule encoding an AGL1/5-like gene product into the vascularplant to produce a transgenic vascular plant characterized by enhancedlignification. The exogenous nucleic acid molecule encoding anAGL1/5-like gene product can be operatively linked to an exogenousregulatory element, which can be a constitutive regulatory element ortissue-selective regulatory element. Tissue-selective regulatoryelements useful in producing a transgenic vascular plant characterizedby enhanced lignification include an AGL1 regulatory element or AGL5regulatory element, or a lignified tissue-selective regulatory elementsuch as a fiber-selective regulatory element, xylem-selective regulatoryelement or tracheid selective regulatory element. An AGL1/5-like geneproduct useful in the invention can have substantially the amino acidsequence of an AGL1 ortholog such as Arabdopsis AGL1 (SEQ ID NO:4), orcan have substantially the amino acid sequence of an AGL5 ortholog suchas Arabdopsis AGL5 (SEQ ID NO:6).

The invention additionally provides methods of enhancing lignificationin a vascular plant by suppressing AGL8-like gene product expression inthe vascular plant, whereby lignification is enhanced.

Further provided by the invention is a transgenic vascular plantcharacterized by enhanced lignification, containing an ectopicallyexpressed nucleic acid molecule including a lignified tissue-selectiveregulatory element operatively linked to a nucleic acid moleculeencoding an AGL1/5-like gene product. In a transgenic vascular plant ofthe invention characterized by enhanced lignification, the AGL1/5-likegene product can have substantially the amino acid sequence of an AGL1ortholog such as Arabdopsis AGL1 (SEQ ID NO:4), or substantially theamino acid sequence of an AGL5 ortholog such as Arabdopsis AGL5 (SEQ IDNO:6), and the lignified tissue-selective regulatory element can be, forexample, a fiber-selective regulatory element, xylem-selectiveregulatory element or a tracheid selective regulatory element. Tissuesderived from a transgenic vascular plant of the invention characterizedby enhanced lignification also are provided.

The invention also provides kits for producing a transgenic vascularplant characterized by altered lignification. Such kits contain anucleic acid molecule including a lignified tissue-selective regulatoryelement and a nucleic acid molecule encoding an AGL8-like gene product,AGL1-like gene product or AGL5-like gene product. Lignifiedtissue-selective regulatory elements useful in a kit of the inventioninclude xylem-selective regulatory elements, tracheid-selectiveregulatory elements, and fiber-selective regulatory elements.

The term “enhanced,” as used herein in reference to lignification in anon-naturally occurring vascular plant of the invention, means asignificantly increased extent of lignification in one or more tissuesas compared to the extent of lignification in a corresponding wild typeplant. Thus, the term “enhanced” is used broadly to encompass bothlignification that is significantly elevated in a tissue or region inwhich lignification normally occurs and the presence of lignification ina tissue or region, which, in a wild type plant, is normally notlignified. The term “enhanced” also encompasses lignification that issignificantly elevated in at least one tissue, although wild type levelsof lignification can be present elsewhere in the plant. One skilled inthe art understands that the term “enhanced” refers to a steady statelevel of lignification and encompasses both increased synthesis anddecreased degradation of lignins.

It is recognized that there can be natural variation in the extent oflignification within a plant species or variety. However, “enhanced”lignification in a non-naturally occurring vascular plant of theinvention readily can be identified by sampling a population of thenon-naturally occurring vascular plants and determining that the extentof lignification is significantly increased, on average, as compared tothe normal distribution of lignification in a population of thecorresponding wild type plant species or variety. Thus, production ofnon-naturally occurring vascular plants of the invention provides ameans to skew the normal distribution of the extent of lignificationsuch that, on average, lignification is significantly greater than in acorresponding wild type plant.

As used herein, the term “AGL1/5-like gene product” means a gene producthaving substantially the amino acid sequence of an AGL1 ortholog orsubstantially the amino acid sequence of an AGL5 ortholog orsubstantially the amino acid sequence of a gene that is a commonancestor of AGL1 or AGL5. An AGL1/5-like gene product is characterized,in part, in that it is a positive regulator of lignification. AnAGL1/5-like gene product also is characterized, in part, by having anamino acid sequence with at least about 30%, 40%, 50%, 60%, 70%, 80%,90% or 95% amino acid identity with SEQ ID NO:4 or SEQ ID NO:6.Arabdopsis AGL1 (SEQ ID NO:3) and Arabdopsis AGL5 (SEQ ID NO:5) areexamples of an AGL1/5-like gene product as defined herein.

One skilled in the art understands that ectopic expression is of a levelsufficient to produce enhanced lignification. Such a level of ectopicexpression is achieved through use of a strong promoter and can also beachieved, for example, by multiple transgene integrations, or throughectopic expression of distinct gene products such as an AGL1 orthologtogether with an AGL5 ortholog. As disclosed in Example IV, one35S::AGL5 transgenic line did not display enhanced lignification, whileboth 35S::AGL1 and 35S::AGL1 35S::AGL5 lines showed enhancedlignification with the later line showing the most dramaticlignification. The results with the 35S::AGL1 35S::AGL5 line support arole for AGL5 in enhancing lignification when expressed at sufficientlevels.

In view of the results disclosed herein, one skilled in the art furtherunderstands that altering the expression of additional combinations oflignification regulatory factors can be useful in reducing or enhancinglignification as desired. For example, ectopic expression of AGL8 can beused in combination with suppression of AGL1 and AGL5 and, if desired,in combination with suppression of R-like bHLH to reduce lignificationin a vascular seed plant. Similarly, ectopic expression of anycombination of AGL1, AGL5 and R-like bHLH can be used to enhancelignification in a vascular seed plant, and such ectopic expression canbe combined, if desired, with suppression of AGL8 expression.

Methods of producing a non-naturally occurring plant characterized byenhanced lignification also are provided herein. Such methods entailectopically expressing a nucleic acid molecule encoding an AGL1/5-likegene product in the vascular plant, whereby lignification is enhanceddue to ectopic expression of the nucleic acid molecule. In oneembodiment, the method entails introducing an exogenous ectopicallyexpressible nucleic acid molecule encoding an AGL1/5-like gene productinto the vascular plant, whereby lignification is enhanced due toectopic expression of the nucleic acid molecule.

The invention also relates to the use of R-like bHLH transcriptionfactors such as the Arabidopsis transcription factor SEQ ID NO:25. Theinvention provides methods of enhancing lignification in a vascularplant by ectopically expressing a nucleic acid molecule encoding anR-like bHLH gene product in the vascular plant, where lignification isenhanced due to ectopic expression of the nucleic acid molecule. In amethod of the invention, the R-like bHLH gene product can havesubstantially the amino acid sequence of an R-like bHLH ortholog such asSEQ ID NO:25. Such methods can be particularly useful for enhancinglignification in woody plants such as trees produced for directutilization as fuel.

In one embodiment, the invention provides a method of enhancinglignification by introducing an exogenous nucleic acid molecule encodinga R-like bHLH gene product into a vascular plant to produce a transgenicvascular plant characterized by enhanced lignification. The exogenousnucleic acid molecule encoding a R-like bHLH gene product can beoperatively linked to an exogenous regulatory element such as aconstitutive regulatory element or tissue-selective regulatory element.

The invention also provides a transgenic vascular plant characterized byenhanced lignification, which contains an ectopically expressed nucleicacid molecule including a heterologous regulatory element operativelylinked to a nucleic acid molecule encoding a R-like bHLH gene product.The encoded R-like bHLH gene product can have substantially the aminoacid sequence of a R-like bHLH ortholog such as the Arabdopsis orthologSEQ ID NO:25.

In one embodiment, the invention provides methods of using a nucleicacid molecule encoding a R-like bHLH ortholog. As used herein, the term“R-like bHLH ortholog” means an ortholog of Arabdopsis R-like bHLH (SEQID NO:25) and refers to a R-like bHLH gene product that, in a particularplant variety, has the highest percentage homology at the amino acidlevel to Arabdopsis R-like bHLH (SEQ ID NO:25). Such a R-like bHLHortholog can be, for example, a Eucalyptus ortholog or an alfalfaortholog. Novel R-like bHLH orthologous cDNAs can be identified fromdatabases or isolated from additional plant species using a nucleotidesequence as a probe and methods well known in the art of molecularbiology as described above.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Production of a 35S::AGL8 Transgenic Arabdopsis PlantDisplaying Reduced Lignification

This example describes methods for producing a transgenic Arabdopsisplant characterized by reduced lignification due to constitutive AGL8expression.

Full-length AGL8 was prepared by polymerase chain reaction amplificationusing primer AGL8 5-γ (SEQ ID NO:9; 5′-CCGTCGACGATGGGAAGAGGTAGGGTT-3′)and primer OAM14 (SEQ ID NO:10; 5′-AATCATTACCAAGATATGAA-3′), andsubsequently cloned into the SalI and BamHI sites of expression vectorpBIN-JIT, which was modified from pBIN19 to include the tandem CaMV 35Spromoter, a polycloning site and the CaMV polyA signal. Arabidopsis wastransformed using the in planta method of Agrobacterium-mediatedtransformation essentially as described in Bechtold et al., C.R. Acad.Sci. Paris 316:1194-1199 (1993), which is incorporated herein byreference. Kanamycin-resistant lines were analyzed for the presence ofthe 35S-AGL8 construct by PCR using a primer specific for the 35Spromoter and a primer specific for the AGL8 cDNA, which produced twofragments of 850 and 550 bp in the 35S::AGL8 transgenic plants. Thesefragments were absent in plants that had not been transformed with the35S-AGL8 construct.

Lignification was assayed in several 35S::AGL8 Arabdopsis lines bystaining fixed fruit sections with toluidine blue as described in Drewset al., Cell 65:991-1002 (1991)) with minor modifications. Lignifiedcell autofluorescence (Barceló, supra, 1997) was examined with Nomarskioptics. Additional lignin histochemical analyses were performed bystaining sections for two minutes in a solution of 2% phloroglucinol in95% ethanol with subsequent photography in 50% hydrochloric acid.

The results, as seen by toluidine blue staining, demonstrated that thenumber of lignified cells adjacent to the dehiscence zone was reduced inthe 35S::AGL8 transgenic lines as compared to the number of lignifiedcells seen in wild type plants.

The extent of lignification in other tissues, for example, in the plantstems is analyzed as described above using toluidine blue staining. Theresults indicate that lignification in other tissues such as stem isreduced in transgenic plants constitutively expressing AGL8, as comparedto the lignification in wild type plants.

These results indicate that ectopic expression of AGL8 can reducelignification in transgenic Arabidopsis.

EXAMPLE II Production of an Arabdopsis agl1 agl5 Double MutantDisplaying Reduced Lignification

This example describes the production of an agl1 agl5 double mutantdisplaying reduced lignification.

A. Production of an agl5 Mutant by Homologous Recombination

A PCR-based assay of transgenic plants was used to identify targetedinsertions into AGL5 as described in Kempin et al., Nature 389:802-803(1997), which is incorporated herein by reference. The targetingconstruct consisted of a kanamycin-resistance cassette that was insertedbetween approximately 3 kb and 2 kb segments representing the 5′ and 3′regions of the AGL5 gene, respectively. A successfully targetedinsertion produces a 1.6 kb deletion within the AGL5 gene such that thetargeted allele encodes only the first 42 of 246 amino acid residues,and only 26 of the 56 amino acids comprising the DNA-bindingMADS-domain. The recombination event also results in the insertion ofthe 2.5 kb kanamycin-resistance cassette within the AGL5 codingsequence.

750 kanamycin-resistant transgenic lines were produced byAgrobacterium-mediated transformation, and pools of transformants wereanalyzed using a PCR assay as described below to determine if any ofthese primary transformants had generated the desired targeted insertioninto AGL5. A single line was identified that appeared to contain theanticipated insertion, and this line was allowed to self-pollinate topermit further analyses in subsequent generations. Genomic DNA from thehomozygous mutant plants was analyzed with more than four differentrestriction enzymes and by several distinct PCR amplifications, and alldata were consistent with the desired targeting event. The regionsflanking the AGL5 gene also were analyzed to verify that there were nodetectable deletions or rearrangements of sequences outside of AGL5.

The kanamycin-resistance cassette within the AGL5 targeting constructcontains sequences that specify transcription termination such thatlittle or no AGL5 RNA was expected in the homozygous mutant plants.Using a probe specific for the 3′ portion of the AGL5 cDNA, AGL5transcripts were detected in wild-type but not in agl5 mutant plants.These data indicate that the targeted disruption of the AGL5 generepresents a loss-of-function allele.

Characterization of the agl5 line indicated that the phenotype of thistransgenic was not different from wild type Arabidopsis.

The AGL5 knockout (KO) construct was prepared in vector pZM104A, whichcarries the kanamycin-resistance cassette flanked by several cloningsites (Miao and Lam, Plant J. 7:359-365 (1995), which is incorporatedherein by reference). Vector pZM104A also contains the gene encodingβ-glucuronidase (GUS), which allows the differentiation ofnon-homologous from homologous integration events. The 3 kb regionrepresenting the 5′ portion of AGL5 was obtained by PCR amplificationusing primer SEQ ID NO:11 (5′-CGGATAGCTCGAATATCG-3′) and primer SEQ IDNO:12 (5′-AACCATTGCGTCGTTTGC-3′). The resulting fragment was cloned intovector pCRII (Invitrogen), and an EcoRI fragment excised and insertedinto the EcoRI site of pZM104A. The 3′ portion of AGL5 was excised as anXbaI fragment from an AGL5 genomic clone in the vector pCIT30 (Ma etal., Gene 117:161-167 (1992), which is incorporated by reference herein)and inserted into the XbaI site of pZM104A. The resulting plasmid,designated AGL5 KO, was used in Agrobacterium-mediated infiltration ofwild-type Arabdopsis plants of the Columbia ecotype. The knockoutconstruct was derived from Landsberg erecta genomic DNA.

Plants containing a homologous recombination event at the AGL5 genomiclocus were identified as follows. Approximately 750 primary (Tl)kanamycin-resistant transformants were selected, and DNA was extractedfrom individual leaves in pools representing ten plants as described inEdwards et al., Nucleic Acids Research 19:1349 (1991), which isincorporated by reference herein. To identify a pool that contained acandidate targeted disruption, isolated DNAs were subjected to PCRamplification using primer SEQ ID NO:13(5′-GTAATTACCAGGCAAGGACTCTCC-3′), which represents AGL5 genomic sequencethat is not contained within the AGL5 KO construct, and primer SEQ IDNO:14 (5′-GTCATCGGCGGGGGTCATAACGTG-3′), which is specific for thekanamycin-resistance cassette. Amplified products were size fractionatedon agarose gels, and used for standard DNA blotting assays with probe 1.One pool of ten plants revealed the anticipated hybridizing band of thecorrect size, and this pool was subsequently broken down into individualplants. A single (T1) plant was identified that appeared to contain thedesired event, and this plant was allowed to self-pollinate for analysesin subsequent generations. This T1 plant was shown to contain theGUS-reporter gene, indicating that in addition to the putativehomologous integration event, there were independent non-homologousevents. Segregation in the subsequent generations allowed theidentification of plants that no longer contained the GUS-reporter gene,and it was these lines that were used for subsequent analyses.

Plants homozygous for the disruption were identified by PCRamplification using primers SEQ ID NO:15(5′-GAGGATAGAGAACACTACGAATCG-3′) and SEQ ID NO:16(5′-CAGGTCAAGTCAATAGATTC-3′), which yielded a single 1.5 kb product inwild type plants, and a single 2.6 kb product in the mutant. Furtherconfirmation that these plants contained the desired disruption wasobtained by PCR amplification with primers SEQ ID NO:17(5′-CAGAATTTAGTGAATAATATTG-3′) and SEQ ID NO:14, which gave the expectedamplified product in the mutant but no product in wild-type plants.

To confirm that the desired disruption had occurred, a series of genomicDNA blots representing wild-type and homozygous mutant (T4 generation)plants were analyzed. Probe 1 hybridized to the expected 3.9 kb XbaIfragment in wild-type and mutant plants, whereas the 1.3 kb XbaIfragment was present only in wild-type. This same probe hybridized to a6 kb EcoRI fragment in wild-type and to the expected 4.1 and 2.8 kbEcoRI fragments in the mutant. Additional digests with BglII and withHindIII confirmed that the mutant plants contained the desired targetedevent. To confirm that there were no detectable deletions orrearrangements outside the targeted region, genomic DNA blots of wildtype and homozygous mutant plants were further analyzed. Probe 2hybridized in wild-type and mutant DNAs to the expected 2.9 kb XmnIfragment, the 1.5 kb and 0.4 kb HincII fragments, and the 0.6 kb HindIIIfragment. Probe 3 hybridized in wild-type and mutant DNAs to the 9 kbScaI fragment, the 3.9 kb XbaI fragment, and the 1.8 kb NdeI fragments.The faintly-hybridizing bands in the ScaI digests represent fragmentsthat span the insertion site, and are, as expected, different sizes inwild-type and agl5 mutant plants.

RNA blotting analyses were performed as follows. Approximately 6 μg ofpolyA+ RNA was purified using DYNABEADS (Dynal) from wild-type and agl5mutant inflorescences, size fractionated and hybridized using standardprocedures (Crawford et al., Proc. Natl. Acad. Sci. USA 83:8073-8076(1986), which is incorporated herein by reference) using a gel-purified450 bp HindIII-EcoRI fragment from pCIT2242 (Ma et al., supra, 1991)specific for the 3′ end of the AGL5 cDNA. The same filter wassubsequently stripped and re-hybridized with a tubulin-specific probe(Marks et al., Plant Mol. Biol. 10:91-104 (1987), which is incorporatedherein by reference). Hybridization with the tubulin probe verified thatapproximately equal amounts of RNA were present in each lane.

B. Production of an agl1 Mutant

A PCR-based screen was used to identify a T-DNA insertion into the AGL1gene essentially as described in Krysan et al., supra, 1996.

RNA blotting analyses demonstrated that AGL1 RNA was not expressed. Theagl1 mutant displayed essentially a wild type phenotype.

C. Production and Characterization of an agl1 agl5 Double Mutant

agl1 agl5 double mutants were generated by crossing the agl1 and agl5single mutants. RNA blotting experiments of the agl1 agl5 double mutantare performed as described above. The results indicate that neither AGL1nor AGL5 RNA is expressed in the agl1 agl5 double mutant.

Toluidine blue analysis of the agl1 agl5 double mutant showed thatpatches of lignified cells adjacent to the dehiscence zone, whichnormally are present in wild type plants, are absent in agl1 agl5 mutantfruit although lignification of the fifth valve cell layer was notaffected (see FIG. 3). These results were further confirmed bylignin-specific histochemical analysis with phloroglucinol, performed asdescribed above. These results indicate that suppression of AGL1expression combined with suppression of AGL5 expression results in anon-naturally occurring plant variety exhibiting an absence oflignification in certain cells.

EXAMPLE III Production of an agl8 Mutant Arabdopsis Plant DisplayingEnhanced Lignification

This example describes methods for producing a non-naturally occurringplant characterized by enhanced lignification due to suppression of AGL8expression.

A. Production of an agl8 Mutant

A mutation designated ful-1 was identified using large scale insertionalmutagenesis with enhancer and gene trap Ds transposable elements(Sundarsen et al., supra, 1995; Springer et al., Science 268:877-880(1995), each of which is incorporated herein by reference). This systemutilized the maize Ac/Ds transposable elements and the reporter geneGUS. Transposition events were selected and screened for reporter geneexpression patterns and mutant phenotypes. The ful-1 mutant wasidentified in the F3 progeny of an enhancer trap line. Backcrossing towild type Landsberg erecta confirmed that ful-1 is a recessive mutation.

To address whether the ful-1 mutation was caused by insertion of theDs-GUS enhancer trap element (DsE), cosegration between the mutantphenotype and expression of the GUS reporter gene was analyzed. Among atotal of 200 mutant plants all were GUS positive, and one-third of thewild type plants were GUS negative as expected in the case of completelinkage between the GUS reporter and the mutation. Genomic analysisrevealed that the mutant plant carried a single transposed Ds element,as expected.

Sequence analysis showed that the Ds element had inserted into theuntranslated leader of the AGL8 gene. Using a probe from the codingregion, AGL8 mRNA was not detectable in flowers from homozygous ful-1mutant plants on RNA blots. The DsE insertion abolished AGL8 geneexpression, indicating that ful-1 leads to a complete loss of AGL8function. Thus, ful-1 is an agl8 mutant.

B. Characterization of Lignification in an agl8 Mutant

The lignification pattern in agl8 fruits was examined by looking atlignin autofluorescence by methods described above. Whereas wild-typefruits (stage 17) show lignification of valve margin cells adjacent tothe dehiscence zone and of the fifth valve cell layer, agl8 fruitsdisplayed additional ectopic lignification of the internal valvemesophyll layers.

These results indicate that suppression of AGL8 expression can produceplants characterized by enhanced lignification.

EXAMPLE IV Production of 35S::AGL1 and 35S::AGL1 35S::AGL5 TransgenicPlants Displaying Enhanced Lignification

This example describes methods for producing transgenic Arabdopsisplants characterized by enhanced lignification due to constitutiveexpression of AGL1 or constitutive expression of AGL1 and AGL5.

A. Production of Transgenic Plants Expressing AGL1 or AGL5 Under Controlof the CaMV 35S Promoter

Transgenic 35S::AGL1, 35S::AGL5 and 35S::AGL1 35S::AGL5 plants weregenerated as follows. A full length AGL1 cDNA was created by fusing theEcoRI fragments of pCIT2241 and pCIT4219 (Ma et al., supra, 1991). TheAGL1 cDNA was subsequently cloned into the BamH1 site of pCGN18 (Jack etal., Cell 76:703-716 (1994)) such that AGL1 transcription was undercontrol of the viral 35S promoter (Benfey and Chua, supra, 1990).

A full-length AGL5 cDNA was PCR amplified with oligonucleotide primersSEQ ID NO:18 (5′-GGAGATCTGAATTCATCTTCCCATCC-3′) and SEQ ID NO:19(5′-CCGGTACCTCAAACAAGTTGCAGAGGTGGTTGGTCTTGGTTGGAGGAATTCTGATTCGGTTCAAG-3′) using pCIT2242 (Ma et al., supra, 1991) astemplate. After cloning this product into the TA vector (Invitrogen), aBglII/KpnI fragment containing the AGL5 cDNA was cloned into the planttransformation vector pMON530 (Monsanto). In the resulting construct,AGL5 transcription was under control of the 35S promoter. Transgenicplants were selected on kanamycin after Agrobacterium-mediatedtransformation as described above (Bechtold et al., supra, 1993).35S::AGL1 transgenic plants were of the Landsberg erecta ecotype, while35S::AGL5 plants were of the Columbia ecotype.

35S::AGL1 and 35S::AGL5 plants were crossed to each other; in the F1generation, 35S::AGL1 35S::AGL5 plants were identified by polymerasechain reaction genotyping using the AGL1 transgene-specificoligonucleotides SEQ ID NO:20 (5′-GAAGGTGGGA GTAGTCACGAC-3′) and SEQ IDNO:21 (5′-CGGAAGGAGGGTTGACGGCA-3′) and the AGL5 transgene-specificoligonucleotides SEQ ID NO:22 (5′-GGTGGTGCGAGTAATGAAGTA-3′) and SEQ IDNO:23 (5′-TGGTCGGAGGGTTAACGGCG-3′).

B. Characterization of Lignification in 35S::AGL1, 35S::AGL5, and35S::AGL1 35S::AGL5 Transgenic Plants

Lignification was characterized in Arabidopsis fruits that ectopicallyexpress AGL1, AGL5 or both. Lignification of 35S::AGL5 fruits appearedidentical to that of wild type fruits. However, 35S::AGL1 and 35S::AGL5fruits (stage 17) displayed ectopic lignification of the valve mesophylllayers, with the most extensive lignification apparent in 35S::AGL135S::AGL5 fruits. Cells at positions corresponding to the mesophylllayers are lignified at the valve margin of wild type fruits, indicatingthat ectopic lignification of 35S::AGL1 35S::AGL5 valve mesophyll layersis a consequence of an acquired valve margin cell identity. The ectopiclignification of the valve mesophyll layers was similar to that seen inagl8 mutants.

These results indicate that a plant ectopically expressing AGL1, orectopically expressing AGL1 and AGL5, displays enhanced lignification.

EXAMPLE V Characterization of agl8 agl1 agl5 Triple Mutants

This example describes analysis of a agl8 agl1 agl5 triple mutant. agl1agl5 plants were crossed to agl8 mutant plants, and the triple mutantsexamined in the F3 generation. In addition, agl1 agl5 GT140 plants werecrossed to agl8 mutants, and agl1 agl5 agl8 GT140 plants examined in theF3 generation.

Fruits from the agl1 agl5 agl8 triple mutant were quite similar inappearance to fruits from the agl8 single mutant, indicating that AGL8activity is largely epistatic to that of AGL1 and AGL5. Ectopiclignification was observed in the fruit valves of the triple mutant,although it appears less extensive than that observed in agl8 mutantvalves. In addition, the GT140 marker displayed a qualitatively weakerbut still expanded domain of expression in the triple mutant.

These results indicate that, while AGL1 and AGL5 can promotelignification of valve margin cells adjacent to the dehiscence zone, andR-like bHLH expression at the valve margin, AGL1 and AGL5 are notabsolutely required for either of these functions. AGL8 can directlyrepress R-like bHLH or other genes promoting lignification of valvemargin cells, or can repress an additional factor that is responsiblefor activating the R-like bHLH transcription factor in the valve margin.

EXAMPLE VI Characterization of R-Like bHLH Expression in Plants withAltered Lignification

This example describes expression of the valve margin molecular markerR-like bHLH in an agl1 agl5 double mutant and in plants ectopicallyexpressing AGL1, AGL5, or both.

The GT140 line contains a GUS reporter inserted adjacent to a molecularmarker specific to the valve margin, the R-like basic helix-loop-helixtranscription factor (R-like bHLH) (Sundaresan et al., supra, 1995);characterization of GUS expression therefore provides a means forfollowing valve margin cell identity when the GT140 line is crossed tomutant or transgenic plants displaying altered lignification.

A. Characterization of R-like bHLH Expression in agl8 Mutant Plants

GT140 plants were crossed to agl8 mutant plants characterized byenhanced lignification. As shown in FIG. 6, R-like bHLH expression asindicated by GUS expression appeared in all valve cell layers of agl8fruits (stage 17), and this expanded domain of expression occurred assoon as the marker was first apparent (stage 13). The expanded domain ofR-like bHLH into the valves of agl8 mutant fruits indicates that agl8valves have acquired the fate of valve margin cells adjacent to thedehiscence zone.

B. Characterization of R-Like bHLH Expression in 35S::AGL1 35S::AGL5Plants

In contrast to its expression in stripes at the valve margin ofwild-type fruits, in 35S::AGL1 35S::AGL5 GT140 fruit, GUS expression wasobserved throughout the valves, indicating that R-like bHLH wasectopically expressed in the valves. These results indicate that in35S::AGL1 35S::AGL5 fruit, the valve layers have acquired the identityof valve margin cells adjacent to the dehiscence zone.

C. Characterization of R-Like bHLH Expression in agl1 agl5 Mutant Plants

GT140 plants were crossed to agl1 agl5 mutant plants, and GUS expressionanalyzed. GUS expression was dramatically altered in agl1 agl5 fruitsdue to the altered fate of agl1 agl5 valve margins. In wild type fruits,GUS was expressed in stripes at the valve margin and in a diffuse domainat the valve base. In contrast, transverse sections of the agl1 agl5fruits showed that GUS was largely absent from cells at the valvemargin, although a low level of expression remained at the base of thevalves.

Together, these results indicate that AGL1, AGL5, or both can positivelyregulate expression of the R-like bHLH transcription factor and thatthis transcription factor can itself be a positive regulator oflignification. These results further indicate that ectopic expression ofR-like bHLH can result in enhanced lignification and that suppression ofR-like bHLH in a vascular seed plant can result in reducedlignification.

All journal article, reference, and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference.

Although the invention has been described with reference to the examplesabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention. Accordingly, theinvention is limited only by the following claims.

1. A method of reducing lignification in cells adjacent to thedehiscence zone in a vascular plant, comprising introducing into thevascular plant a plant promoter operably linked to an exogenous nucleicacid that encodes a polypeptide at least 95% identical to SEQ ID NQ:25to reduce expression of an endogenous bHLH gene product, therebyreducing lignification.
 2. A transgenic vascular plant characterized byreduced lignification in cells adjacent to the dehiscence zone, thetransgenic vascular plant comprising a plant promoter onerably linked toan exogenous nucleic acid that encodes a fragment of SEQ ID NO:25 toreduce expression of an endogenous bHLH gene product, thereby reducinglignification.
 3. A method of reducing lignification in cells adjacentto the dehiscence zone in a vascular plant, comprising introducing intothe vascular plant a plant promoter operably linked to an exogenousnucleic acid that encodes a fragment of SEQ ID NO:25 to reduceexpression of an endogenous bHLH gene product, thereby reducinglignification.
 4. The method of claim 1, wherein the endogenous bHLHgene product comprises SEQ ID NO:25.
 5. The method of claim 1, whereinthe plant promoter is operatively linked to the exogenous nucleic acidin the sense orientation, thereby suppressing expression of theendogenous bHLH gene product.
 6. The method of claim 1, wherein theplant promoter is operatively linked to the exogenous nucleic acid inthe antisense orientation, thereby suppressing expression of theendogenous bHLH gene product.
 7. The method of claim 1 or claim 3,wherein said vascular plant is a woody plant.
 8. The method of claim 7,wherein said woody plant is selected from the group consisting ofEucalyptus, cottonwood, alder, Douglas fir, Hemlock, pine and spruce. 9.The method of claim 1 or claim 3, wherein said vascular plant is aleguminous plant.
 10. The method of claim 9, wherein said leguminousplant is selected from the group consisting of alfalfa, clover, lucerne,birdsfoot trefoil, Stylosanthes, Lotononis bainessii and sainfoin. 11.The method of claim 1 or claim 3, wherein said vascular plant is aforage grass.
 12. The method of claim 11, wherein said grass is selectedfrom the group consisting of bahiagrass, bermudagrass, dallisgrass,pangolagrass, big bluestem, indiangrass, switchgrass, smooth bromegrass,orchardgrass, timothy, Kentucky bluegrass and tall fescue.
 13. Thetransgenic plant of claim 2, wherein the promoter is operatively linkedto the exogenous nucleic acid in the sense orientation.
 14. Thetransgenic plant of claim 2, wherein the promoter is operatively linkedto the exogenous nucleic acid in the antisense orientation.
 15. A tissuederived from a transgenic plant of claim
 2. 16. The method of claim 3,wherein the plant promoter is operatively linked in the senseorientation to the polynucleotide that encodes a fragment of SEQ IDNQ:25, thereby suppressing expression of the endogenous bHLH geneproduct.
 17. The method of claim 3, wherein the plant promoter isoperatively linked in the antisense orientation to the polynucleotidethat encodes a fragment of SEQ ID NO:25, thereby suppressing expressionof the endogenous bHLH gene product.